ES2581318T3 - Simultaneous and integrated selection and evolution of antibody / protein performance and expression in production hosts - Google Patents

Abstract

Method of evolution and expression of an antibody in a eukaryotic cell production host; Understanding the method: a. select a template antibody; b. evolve the template antibody to allow the production of a set of mutant antibodies in a eukaryotic cell production host; C. examine the mutant antibodies to determine at least one predetermined property, characteristic or activity; d. selecting a higher mutant antibody from the set of mutant antibodies based on the optimization of the at least one predetermined property, characteristic or activity compared to the same property, characteristic or activity of the template antibody; and e. express the higher mutant antibody in the same eukaryotic cell production host as in step (b) for any commercial scale.

Description

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DESCRIPTION

Simultaneous and integrated selection and evolution of performance and expression of antibodies / proteins in production hosts.

Field of the Invention

In a particular aspect, the present invention is relevant for antibodies and for their optimization by evolution of antibodies. Therapeutic antibodies are discovered, evolved and manufactured in the same host using the same genetic systems.

Background of the invention

A variety of antibody and other protein systems that generate protein therapeutic molecules have been designed, developed and implemented. More recently, many evolution systems have been developed to enhance the function of proteins. Separately, mammalian expression systems have been developed for the production of high-performance antibodies and other proteins for therapeutic applications. To date, no group has developed a system to allow the generation, evolution of an antibody or protein, and production / manufacture of proteins in an individual effective mammalian expression system.

WO 2002092780 relates to methods for generating sets, or libraries, of nucleic acids encoding antigen binding sites, such as antibodies, antibody domains or other fragments. The method is to generate variant antigen binding sites by altering template nucleic acids including saturation mutagenesis, synthetic linkage reassembly, or a combination thereof.

Rajpal et al., "A general method for greatly improving the affinity of antibodies by using combinatorial libraries", Proc Natl Acad Sci U S A., vol. 102, pages 8466-8471, (2005) disclose a method to improve antibody affinity using the so-called revised mutagenesis that simultaneously evaluates and optimizes combinatorial amino acid mutations selected in the antibody. The procedure focuses on a precise distribution within one or more domains of the region determining complementarity and explores the synergistic contribution of the amino acid side chain chemistry.

Iba et al., "Expression vectors for the introduction of highly diverged sequences into the six complementitydetermining regions of an antibody", Gene, vol. 194, pages 35-46, (1997) disclose three kinds of phagemid vectors that allow the simultaneous introduction of sequences with high divergence in six regions determining the complementarity of an antibody by polymerase chain reaction with oligodeoxynucleotide primers degenerated The phages expressed either the form of Fv, single chain Fv (scFv) or Fab of an Ac fused with the cplll half molecule on the surface of phage M13.

WO 2005/003345 discloses a method of mutagenesis by which a predetermined amino acid is introduced into each and every position of a selected set of positions in a preselected region (or several different regions) of a polypeptide to produce a library of polypeptide analogs. The libraries produced can be used to study the role of specific amino acids in the structure and function of the polypeptide and to develop new or improved polypeptides such as antibodies, antibody fragments, single chain antibodies, enzymes and ligands.

WO 2003/023032 discloses a process for the directed evolution of high yield of peptides and proteins, particularly those acting in complex biological environments. The proteins and peptides include, but are not limited to, intracellular proteins, messenger / signaling / hormone proteins, and viral proteins.

Many antibodies are developed using phage display systems of bacteria in bacteria, while the expression of full-length antibodies is carried out primarily in mammalian cells. This lack of similarity makes the evolution or selection of clones for expression impossible. Other barriers include the traditional requirement that large numbers of variants be examined using traditional technologies and high-level mammalian expression systems have been optimized for expression, not the donation of large numbers of variants. An integrated antibody / protein selection, evolution and mammalian expression system has not been previously designed. The use of eukaryotic cells to handle large numbers, combined with non-stochastic evolution of the antibody inside an optimized eukaryotic host cell, increases the likelihood of success and greatly accelerates the procedure to generate an optimized antibody that would be expressed sufficiently high in eukaryotic cells that you want to make.

Summary of the invention

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The present disclosure provides methods of integrating the generation and / or selection of therapeutic antibodies, evolution and expression in a eukaryotic host, such as a mammalian cell host or a yeast cell host, for manufacturing in an individual system. Therapeutic antibodies are generated, optimized and manufactured in the same eukaryotic host system. The system known as Complete Integrated Antibody Optimization (CIAO! ™) allows the simultaneous evolution of optimization of antibody expression and performance.

In one embodiment, the disclosure provides a method of selection, evolution and expression of an antibody in a eukaryotic cell production host; the method comprising the steps of: selecting a template antibody; evolve the template antibody to produce a set of mutant antibodies in the eukaryotic cell production host; examine the mutant antibodies to determine the at least one predetermined property, characteristic or activity; selecting a superior mutant antibody from the set of mutant antibodies based on the optimization of the at least one predetermined property, characteristic or activity compared to the same property, characteristic or activity of the template antibody; and expressing the superior mutant antibody in the same eukaryotic cell production host that is used in the stage of making it evolve for any commercial scale. In one aspect, the selection step comprises generating a library of anti-antigen antibodies in a eukaryotic cell production host with presentation on the antibody cell surface; examine the library to determine at least one predetermined property, characteristic or activity; and select a template antibody from the library. In another aspect, the library of anti-antigen antibodies is a library of humanized anti-antigen antibodies.

In one aspect, the eukaryotic cell production host is selected from 3T3 mouse fibroblast cells; BHK21 Syrian Hamster Fibroblast Cells; dog epithelial cells, MDCK; Hela human epithelial cells; Kangaroo rat epithelial cells PtK1; SP2 / 0 mouse plasma cells; and NS0 mouse plasma cells; human embryonic kidney cells HEK 293; monkey monkey kidney cells; Chinese hamster ovary cells CHO, CHO-S; R1 mouse embryonic cells; E14.1 mouse embryonic cells; human embryonic cells H1; human embryonic cells H9; human embryonic cells PER C.6; S. cerevisiae yeast cells; or Picchia yeast cells. In a particular aspect, the eukaryotic system is CHO-S or HEK293. In one aspect, the examination stages use fluorescence activated cell classification (FACS).

In another aspect, the step of evolving comprises producing a set of mutant antibodies formed from the template antibody having m complementarity determining regions (CDR), in which m is an integer selected from 1, 2, 3, 4, 5 or 6, each of said CDRs comprising n amino acid residues, the method comprising generating mxn independent sets of antibodies, each set comprising member antibodies having a predetermined number of different amino acid residues X at a predetermined individual position of the CDR; in which each set of antibodies differs in the predetermined individual position; and the number of different member antibodies generated is equivalent to m x n x X. In a particular aspect, m is 6.

In one aspect, the step of evolving comprises generating n-1 independent sets of mutant polypeptides from the template antibody, each set comprising member polypeptides having a predetermined number of different amino acid residues at a predetermined individual position of the polypeptide; in which each set of polypeptides differs in the predetermined individual position; and the number of different member polypeptides generated is equivalent to [n-1] x X. In one aspect, X is 19.

In another aspect, the examination step comprises testing each member polypeptide to determine at least one predetermined property, characteristic or activity; identify any change in said property, characteristic or activity of the member polypeptide in relation to the template polypeptide; create a functional map in which the functional map is used to identify positions and mutations in the mutant polypeptide that result in a superior mutant and / or a silent mutation compared to the template polypeptide.

In another aspect, the antibody fragment is selected from a heavy chain, light chain, variable domain, constant domain, hypervariable region, complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and determining region of complementarity 3 (CDR3).

In another aspect, the generation step comprises subjecting a polynucleotide containing codons encoding said template polypeptide to polymerase-based amplification using a degenerate oligonucleotide 64 times for each codon to be mutagenized, in which each of said degenerate oligonucleotides 64 Sometimes it is composed of a first homologous sequence and a degenerate N, N, N triplet sequence, so that a set of progeny polynucleotides is generated; and subjecting said set of progeny polynucleotides to clonal amplification so that polypeptides encoded by the progeny polynucleotides are expressed.

Also part of the invention is the method according to revindication 1 in which the predetermined property, characteristic or activity is selected from reduction of antibody-protein aggregation, enhancement of antibody stability, increase in antibody solubility, introduction of sites of glycosylation,

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introduction of conjugation sites, reduction of immunogenicity, potentiation of antibody expression, increase of antigenic affinity, decrease of antigenic affinity, change in binding affinity, change in immunogenicity or enhancement of specificity.

In another aspect, the stage of evolving comprises an evolution technique selected from one of complete position evolution (CPE); evolution of full position insertion (CPI); evolution of complete position deletion (CPD); complete position evolution (CPE) followed by combinatorial protein synthesis (CPS); complete position deletion evolution (CPD) followed by combinatorial protein synthesis (CPS); or evolution of complete position deletion (CPD) followed by combinatorial synthesis of proteins (CPS).

The antibody is a full length antibody.

In another aspect, the at least one predetermined property, characteristic or activity in the examination stage (e) comprises one or more of (1) examining for a silent mutation and (2) examining for a change of sense mutation; in comparison with the template antibody.

In another aspect, one or more parts of the antibody selected from Fc and Fv; framework region; and one or more CDRs are modified in the upper mutant human antibody compared to the human template antibody that is to be evolved.

In another aspect, the examination step comprises examining the set of mutant human antibodies to determine the at least one predetermined property, characteristic or activity and examining to simultaneously determine the modified expression.

In another aspect, the host of cellular production is a host of eukaryotic production and the step of evolving comprises making the human antibody mold evolve to produce a set of mutant human antibodies in the host of eukaryotic cellular production with presentation on the cell surface.

In another embodiment, the disclosure provides a method of evolution for the enhanced expression and fabrication of a human antibody in a eukaryotic cell production host; the method comprising selecting a human antibody template for evolution; evolve the human template antibody comprising the generation of mutant codons encoding the template human antibody to produce a set of mutant human antibodies in the production host; examine the set of mutant human antibodies to determine at least one predetermined property, characteristic or activity and examine to determine the enhanced expression compared to the human template antibody; selecting a superior mutant human antibody from the set of mutant human antibodies based on (1) retention or optimization of the at least one predetermined property, characteristic or activity and (2) enhanced expression compared to the template human antibody; and manufacturing the superior mutant human antibody comprising expressing the superior mutant human antibody in the same production host as in the stage of evolving.

In one aspect, the mutant codons of the upper mutant human antibody result in at least one silent mutation and / or sense change mutation. In another aspect, the mutant codons of the upper mutant human antibody result in at least one silent mutation.

In a further aspect, the template human antibody is an antibody therapeutic drug approved by an ethical committee, and the superior mutant human antibody is a biosimilar drug.

In another aspect, the selection step comprises selecting a higher mutant human antibody from the set of mutant human antibodies based on (1) optimization of the at least one predetermined property, characteristic or activity and (2) enhanced expression compared to the human antibody mold.

In another embodiment, the disclosure provides a method of identification and production of a target human antibody, the method comprising generating a library of human antibodies in a eukaryotic cell production host with presentation on the cell surface of antibodies; examine the library to determine at least one predetermined property, characteristic or activity; identify a target human antibody from the library based on the at least one predetermined property, characteristic or activity; and expressing the target human antibody in the same eukaryotic cell production host as in the generation step to produce a target human antibody. In one aspect, the target human antibody is a full length antibody.

In another embodiment, the disclosure provides a method of evolving a human antibody in a manufacturing host, the method comprising mutating a human antibody template to produce a set of mutant human antibodies in a manufacturing host; and examine the set of mutant progeny antibodies to determine at least one predetermined property, characteristic or activity. In one aspect, the method comprises

also select a superior mutant human antibody from the set of mutant human antibodies based on the at least one predetermined property, characteristic or activity. In another aspect, the method further comprises manufacturing the upper mutant human antibody which comprises expressing the upper mutant human antibody in the same production host as in the mutation stage. In another aspect, the step of selection further comprises selecting a superior mutant human antibody from the set of mutant human antibodies based on (1) optimization of the at least one predetermined property, characteristic or activity in comparison to the template human antibody, and ( 2) modified expression compared to the human antibody template. In one aspect, the modified expression is enhanced expression.

In another embodiment, the disclosure provides a method of evolution and fabrication of a human antibody in a host of cellular manufacturing; the method comprising mutating a template human antibody to produce a set of mutant human antibodies in a manufacturing host; examine the set of mutant human antibodies to determine at least one predetermined property, characteristic or activity and examine for modified expression; selecting a higher mutant human antibody from the set of mutant human antibodies based on (1) optimization of the at least one predetermined property, characteristic or activity, and (2) modified expression compared to the template human antibody; and manufacturing the human antibody comprising expressing the superior mutant human antibody in the same manufacturing host as in the mutation stage.

In a further embodiment, the disclosure provides a method of evolution for the enhanced expression and manufacture of a human antibody in a eukaryotic cell production host; method 20 comprising mutating a human template antibody comprising generation of mutant codons encoding the template human antibody to produce a set of mutant human antibodies in a manufacturing host; examine the set of mutant human antibodies to determine at least one predetermined property, characteristic or activity and examine to determine the enhanced expression compared to the human template antibody; selecting a superior mutant human antibody from the set of mutant human antibodies based on (1) retention or optimization of the at least one predetermined property, characteristic or activity and (2) enhanced expression compared to the template human antibody; and manufacturing the superior mutant human antibody in the same manufacturing host as in the mutation stage.

In one aspect, the examination step comprises examining the set of mutant human antibodies to determine the at least one predetermined property, characteristic or activity and examining to determine the simultaneously enhanced expression.

Brief description of the drawings

Figure 1 provides an outline of the CIAO ™ method of integration of and / or selection of therapeutic antibodies, evolution and expression in a eukaryotic host, such as a mammalian cell host or a yeast cell host, for manufacturing in An individual system.

35 Figure 2 illustrates how complete position evolution (CPE ™) is used to generate a specific molecule database (EvoMap ™).

Figure 3 shows a complete position synthesis scheme (CPS ™) that can be used to combine higher CPE ™ mutants.

Figure 4 shows a diagram of the evolution of full position insertion (CPI ™).

Figure 5 illustrates a combination of evolution methods: a prolonged nucleic acid from evolution CPI ™ undergoes full position evolution (CPE ™) and is used to generate a specific molecule database (EvoMap ™).

Figure 6 shows a complete position deletion evolution (CPD ™) scheme.

Figure 7 illustrates another combination of evolution methods: a shortened nucleic acid from evolution 45 CPD ™ undergoes full-position evolution (CPE ™) and is used to generate a specific molecule database (EvoMap ™).

Definition of terms

To facilitate the understanding of the examples provided herein, certain methods and / or terms that appear frequently will be described.

The term "agent" is used herein to indicate a polypeptide, a mixture of polypeptides, a

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alignment of spatially located compounds (eg, a VLSIPS peptide alignment, polynucleotide alignment and / or a combinatorial small molecule alignment), biological macromolecule, a bacteriophage peptide presentation library, a bacteriophage antibody (eg scFv ) presentation library, a presentation library of polysomal peptides, or an extract produced from biological materials such as bacteria, plants, fungi or cells or tissues of animals (particularly mammals). Agents are evaluated to determine their possible activity as antineoplasics, anti-inflammatories or modulators of apoptosis by their inclusion in test tests described below in this document. Agents are evaluated to determine their possible activity as specific inhibitors of the interaction of proteins (i.e., an agent that selectively inhibits a binding interaction between two predetermined polypeptides but does not substantially interfere with cell viability) by inclusion in assays of Examination described below in this document.

The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (-COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds. "Twenty alpha-amino acids that form naturally encoded polypeptides" are understood in the art and refer to: alanine (wing or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cystel (cys or C), glutamic acid (glu or E), glutamine (gln or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (trp or W ), tyrosine (tyr or Y), and valine (val or V).

The term "amplification" means that the number of copies of a polynucleotide increases.

The term "antibody", as used herein, refers to intact immunoglobulin molecules, as well! as to fragments of immunoglobulin molecules, such as Fab, Fab ', (Fab') 2, Fv, and SCA fragments, which can bind to an epltope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen (eg, a polypeptide antigen) of the antibody from which they are derived, can be produced using methods well known in the art (see, for example, Harlow and Lane, cited above), and are described further, as follows. The antibodies can be used to isolate preparative amounts of the antigen by immunoaffinity chromatography. Other various uses of such antibodies are to diagnose and / or stage the disease (for example, neoplasia) and for therapeutic application to treat a disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients.

A Fab fragment consists of a monovalent antigen binding fragment of an antibody molecule, and can be produced by digestion of a complete antibody molecule with the papal enzyme, to provide a fragment consisting of an intact light chain and a part of a heavy chain

A Fab 'fragment of an antibody molecule can be obtained by treating a complete antibody molecule with pepsin, followed by reduction, to provide a molecule consisting of an intact light chain and a part of a heavy chain. Two Fab 'fragments are obtained per antibody molecule treated in this way.

A fragment (Fab ') 2 of an antibody can be obtained by treating a complete antibody molecule with the enzyme pepsin, without further reduction. A fragment (Fab ') 2 is a dimer of two Fab' fragments, held together by two disulfide bonds.

An Fv fragment is defined as a fragment modified by genetic engineering that contains the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

A single chain antibody ("SCA") is a single chain molecule modified by genetic engineering that contains the variable region of a light chain and the variable region of a heavy chain, joined by a suitable flexible polypeptide linker.

The term "biosimilar drug", also called "continuation biological drug", refers to new officially approved versions of innovative biopharmaceutical products, after the expiration of a patent or exclusivity.

The term "host of cellular production," or "host of fabrication," refers to a cell line used for the production or manufacture of antibodies. Eukaryotic cells such as mammalian cells, including, but not limited to human, mouse, hamster, rat, monkey ace! as cell lines of yeasts, insects and plants. Prokaryotic cells can be used. In one aspect, a mammalian cell production host is selected from a member of the group consisting of 3T3 mouse fibroblast cells; BHK21 Syrian Hamster Fibroblast Cells; dog epithelial cells, MDCK; Hela human epithelial cells; epithelial cells of

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Kangaroo rat PtK1; SP2 / 0 mouse plasma cells; and NS0 mouse plasma cells; human embryonic kidney cells HEK 293; monkey monkey kidney cells; Chinese hamster ovary cells CHO, CHO-S; R1 mouse embryonic cells; E14.1 mouse embryonic cells; human embryonic cells H1; human embryonic cells H9; human embryonic cells PER C.6. In another aspect, the host of cellular production is to GS-NS0 or GS-CHOK1 cell line. In another aspect, the host of cellular production is selected from S. cerevisiae yeast cells; and Picchia yeast cells.

A molecule that has a "chimeric property" is a molecule that is: 1) partly homologous and partly heterologous to a first reference molecule; while 2) at the same time it is partly homologous and partly heterologous to a second reference molecule; without 3) excluding the possibility of being at the same time partly homologous and partly heterologous to one or more additional reference molecules. In a non-limiting embodiment, a chimeric molecule can be prepared by assembling a rearrangement of partial molecular sequences. In a non-limiting aspect, a chimeric polynucleotide molecule can be prepared by synthesizing the chimeric polynucleotide using a plurality of molecular molds, so that the resulting chimeric polynucleotide has the properties of a plurality of molds.

The term "related" as used herein refers to a genetic sequence that is evolutionarily and functionally related between species. For example, but without limitation, in the human genome the human CD4 gene is the gene related to the mouse 3d4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein It works in signaling the activation of T cells through recognition of CMH-restricted class II antigen.

The term "commercial scale" means the production of an antibody at an appropriate scale for resale.

A "comparison window," as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions in which a polynucleotide sequence can be compared to a reference sequence of at least 20 contiguous nucleotides. and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less compared to the reference sequence (which does not include additions or deletions) for Optimal alignment of the two sequences. Optimal sequence alignment to align a comparison window can be carried out using the local homolog algorithm of Smith and Waterman (1981) Adv. Appl. Math 2: 482 by the homology alignment algorithm of Needlemen and Wuncsch J. Mol. Biol. 48: 443 (1970), by means of the similarity search method of Pearson and Lipman Proc. Natl Acad. Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics version 7.0 software package, Genetics Computer Group, 575 Science Dr., Madison, Wis. ), or by inspection, and the best alignment is selected (that is, which results in the highest percentage of homologation throughout the comparison window) generated by the various methods.

As used herein, the term "complementarity determining region" and "CDR" refer to the term recognized in the art as exemplified by Kabat and Chothia. The CDR definitions are also generally known as supervariable regions or hypervariable loops (Chothia and Leks, 1987; Clothia et al., 1989; Kabat et al., 1987; and Tramontano et al., 1990). Variable region domains typically comprise the approximately 105-115 amino-terminal amino acids of a naturally occurring immunoglobulin chain (eg, amino acids 1-110), although somewhat shorter or longer variable domains are also suitable for form single chain antibodies. The CDRs are part of immunoglobulins that determine the specificity of these molecules and come into contact with a specific ligand. CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions, cDR1, CDR2 and CDR3, in each V domain. CDR-H represents a CDR region of a variable heavy chain and CDR-L refers to a CDR region of a variable light chain. H means the variable heavy chain and L means the variable light chain. The CDR regions of an Ig-derived region can be determined as described in Kabat (1991). Sequences of Proteins of Immunological Interest, 5th edition, NIH publication No. 91-3242 U.S. Department of Health and Social Services, Chothia (1987) J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.

The term "complete" is used herein to refer to an evolution technique in which all possible changes are made in each position of a template polynucleotide or template polypeptide and the polynucleotide or polypeptide is tested to confirm that They have made the intended changes.

"Conservative amino acid substitutions" refers to the ability to exchange waste having similar side chains. For example, a group of amino acids that have aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids that have aliphatic hydroxyl side chains is serine and threonine; a group of amino acids that have amide containing side chains is asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine and histidine; and a group of amino acids that have sulfur-containing side chains is cystel and methionine. Amino acid substitution groups

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Preferred conservatives are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine.

The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (ie, is identically, not strictly evolutionarily related) to all or a part of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contrast, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a part of a reference polynucleotide sequence. For illustration, the "TATAC" nucleotide sequence corresponds to a reference "TATAC" sequence and is complementary to a "GTATA" reference sequence.

As used herein, the term "defined sequence framework region" refers to a set of defined sequences that are selected on a non-random basis, generally based on experimental data or structural data; For example, a defined sequence framework region may comprise a set of amino acid sequences that are predicted to form a lamellar structure or may comprise a repetition motif of leucine zipper heptad, a zinc finger domain, among others. variations A "defined sequence nucleus" is a set of sequences that encompass a limited range of variability. While (1) a completely random decamerican sequence of the 20 conventional amino acids can be any of (20) 10 sequences, and (2) a pseudo-random decamerican sequence of the 20 conventional amino acids can be any of (20) 10 sequences but present a bias towards certain residues in certain positions and / or in general, (3) a nucleus of defined sequences is a subset of sequences if each residue position was allowed to be any of the 20 admissible conventional amino acids (and / or amino / imino acids not admissible conventional). A core of defined sequences generally comprises variant and invariant residue positions and / or comprises variant residue positions that may comprise a residue selected from a defined subset of amino acid residues), and the like, either segmented or throughout the entire length of the individual selected library member sequence. The defined sequence nuclei can refer either to amino acid sequences or to polynucleotide sequences. As an illustration and without limitation, the sequences (NNK) 10 and (NNM) 10, in which N represents A, T, G, or C; K represents G or T; and M represents A or C, they are nuclei of defined sequences.

The term "deimmunization" as used herein refers to the production of a variant of the template binding molecule, which is modified compared to an original natural type molecule making said variant non-immunogenic or less immunogenic in beings. humans. The deimmunized molecules according to the invention refer to antibodies or parts thereof (such as framework and / or CDR regions) of non-human origin. Corresponding examples are antibodies or fragments thereof as described in US 4,361,549. The term "deimmunized" also refers to molecules, which show a reduced propensity to generate epltopos of T cells. According to this invention, the term "reduced propensity to generate epltopos of T cells" refers to the elimination of epltopos from T cells that leads to the activation of specific T cells.

In addition, reduced propensity to generate T-cell epltopos means substitution of amino acids that contribute to the formation of T-cell epltopos, i.e. amino acid substitution, which are essential for the formation of a T-cell epltopo. In other words, reduced propensity. To generate T-cell epltopos refers to reduced immunogenicity or reduced ability to induce antigen-independent proliferation of T-cells. In addition, reduced propensity to generate T cell epltopos refers to deimmunization, which means loss or reduction of possible T cell epltopes of amino acid sequences that induce antigen-independent T cell proliferation.

The term "T-cell epltopo" as used herein refers to short peptide sequences that can be released during the degradation of peptides, polypeptides or proteins inside cells and subsequently presented by major histocompatibility complex (CMH) molecules. to trigger the activation of T cells; see, among others, WO 02/066514. For peptides presented by CMH class II, such activation of T cells can then induce an antibody response by direct stimulation of B cells to produce said antibodies.

"Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that acts only in certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as known to the person skilled in the art. For analytical purposes, 1 mg of plasmid or DNA fragment with approximately 2 units of enzyme in approximately 20 ml of buffer solution is usually used. In order to isolate DNA fragments for plasmid construction, typically 5 to 50 mg of DNA with 20 to 250 units of enzyme are digested in a larger volume. The manufacturer specifies appropriate buffers and substrate amounts for particular restriction enzymes. Usually incubation times of approximately 1 hour are used at 37 ° C, but may vary according to the supplier's instructions. After digestion,

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The reaction is subjected to electrophoresis directly on a gel to isolate the desired fragment.

The term "DNA exchange" is used herein to indicate recombination between substantially homologous but not identical sequences, in some embodiments DNA exchange may involve cross-linking through non-homologous recombination, such as through cer / cer systems. lox and / or flp / frt and the like. DNA exchange can be random or non-random.

As used in this invention, the term "epltopo" refers to an antigenic determinant in an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as a phytase specific antibody, binds. Antigenic determinants usually consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics, as well! as specific face features. As used herein, "epltopo" refers to that part of an antigen or other macromolecule that can form a binding interaction that interacts with the variable region binding body of an antibody. Normally, such binding interaction manifests as an intermolecular contact with one or more amino acid residues of a CDR.

The term "evolution" refers to a change in at least one property, characteristic or activity of a genetically or synthetically modified antibody compared to a template antibody.

The terms "fragment", "derivative" and "analog" when referring to a polypeptide comprise a polypeptide that retains at least one biological function or activity that is at least essentially the same as that of the reference polypeptide. In addition, the terms "fragment," "derivative," or "analog" are exemplified by a "pro-form" molecule, such as a low-activity pro-protein that can be modified by cleavage to produce a mature form with significantly greater activity. .

A method is provided herein to produce from a template polypeptide a set of progeny polypeptides in which a "full range of individual amino acid substitutions" is represented at each amino acid position. As used herein, "full range of individual amino acid substitutions" refers to the naturally encoded alpha-amino acids that form naturally encoded polypeptides, as described herein.

The term "gene" means the segment of DNA involved in the production of a polypeptide chain; It includes regions that precede and follow the coding region (llder and trailer sequences) as! as intermediate sequences (introns) between individual coding segments (exons).

"Genetic instability," as used herein, refers to the natural tendency of highly repetitive sequences to be lost through a process of reducing events that generally involve sequence simplification through the loss of repeated sequences. Deletions tend to involve the loss of a copy of a repetition and everything in between the repetitions.

The term "heterologous" means that a single stranded nucleic acid sequence cannot hybridize with another single stranded nucleic acid sequence or its complement. Therefore, heterologous zones means that zones of polynucleotides or polynucleotides have zones or regions within their sequence that cannot hybridize with another nucleic acid or polynucleotide. Such regions or zones are for example zones of mutations.

The term "homologous" or "homeologist" means that a nucleic acid sequence of single stranded nucleic acid can hybridize with a complementary single stranded nucleic acid sequence. The degree of hybridization may depend on several factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentrations as discussed below. Preferably, the identity region is greater than 5 bp, more preferably the identity region is greater than 10 bp.

The term "humanized" is used to describe antibodies in which complementarity determining regions (CDRs) of a mammalian animal, for example, a mouse, are combined with a human framework region. Often, polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework region (and optionally constant regions) to form polynucleotides encoding complete antibodies (eg, humanized or fully human), fragments of antibody, and the like. In another aspect, in addition to mouse antibodies, other species can be humanized, such as, for example, other rodents, camel, rabbit, cat, dog, pig, horse, cow, fish, llama and shark. In a broad aspect, any species that produces antibodies in the production of humanized antibodies can be used. Additionally, the antibodies of the invention can be chimeric, similar to human, humanized or fully human, to reduce their possible antigenicity, without reducing their affinity for their target. Chimeric, human-like and humanized antibodies have generally been described in the art.

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By incorporating the least amount of foreign sequence possible in the hybrid antibody, antigenicity is reduced. The preparation of these hybrid antibodies can be carried out by methods well known in the art.

In an alternative aspect, human or mouse antibodies are adapted to a different recipient species, such as a threatened species, to provide therapeutic agents for the recipient species while protecting against a negative immune response. In this aspect, the framework regions of a recipient species are used in combination with the CDRs of antibodies of a known species or second species.

A variable region of immunoglobulin light or heavy chain consists of a "framework" region interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and the CDRs has been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat et al., 1987). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. As used herein, a "human framework region" is a framework region that is substantially identical (85 or more, usually 90-95 or more) to the framework region of a human immunoglobulin that is produced from natural way The framework region of an antibody, ie the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. CDRs are primarily responsible for binding to an epltopo of an antigen. According to this invention, a framework region refers to a region in the V domain (VH or VL domain) of immunoglobulins that provides a protein framework for the hypervariable complementarity determining (CDR) determining regions that come into contact with the antigen. In each domain V, there are four framing regions designated as FR1, FR2, FR3 and FR4. The framework region 1 encompasses the region from the N-terminal end of domain V to the beginning of CDR1, the framework region 2 refers to the region between CDR1 and CDR2, the framework region 3 encompasses the region between CDR2 and CDR3 and the framework region 4 means the region from the end of CDR3 to the C-terminal end of domain V; see, among others, Janeway, Immunobiology, Garland Publishing, 2001, 5th ed. Therefore, the framework regions encompass all regions outside the CDR regions in the VH or VL domains.

The person skilled in the art is easily able to deduce the framework regions and the CDRs from a given sequence; see Kabat (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242 U.S. Department of Health and Social Services, Chothia (1987) J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.

The benefits of this invention extend to "industrial applications" (or industrial processes), a term used to include applications in the commercial industry itself (or simply industry) as! as non-commercial industrial applications (for example, biomedical research in a non-profit institution). Relevant applications include those in the areas of diagnosis, medicine, agriculture, manufacturing and teaching.

The term "identics" or "identity" means that two nucleic acid sequences have the same sequence or a complementary sequence. Thus, "identity zones" means that regions or zones of a polynucleotide or the global polynucleotide are identical or complementary to zones of another polynucleotide or the polynucleotide.

The term "isolated" means that the material is removed from its original environment (for example, the natural environment if it occurs naturally). For example, an enzyme or a polynucleotide that is produced naturally or present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from part or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and / or such polynucleotides or enzymes could be part of a composition, and still be isolated because such vector or composition is not part of its natural environment.

By "isolated nucleic acid" is meant a nucleic acid, for example, a DNA or RNA molecule, which is not immediately adjacent to the 5 'and 3' flanking sequences to which it is normally immediately contiguous when present in the genome. that occurs naturally from the organism from which it is derived. The term therefore describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but in a different place from that which occurs naturally); and a nucleic acid that exists as an independent molecule, for example, a DNA fragment produced by PCR amplification or digestion with restriction enzymes, or an RNA molecule produced by in vitro transcription. The term also describes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion polypeptide.

"Ligation" refers to the phosphodiester bond formation process between two double stranded nucleic acid fragments (Maniatis et al., 1982, p. 146). Unless otherwise provided, ligation can be achieved using known conditions and buffers with 10 units of T4 DNA ligase ("ligase") per 0.5 mg of approximately equimolar amounts of the DNA fragments to be ligated.

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As used herein, "linker" or "spacer" refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in a preferred configuration, for example, so that the random peptide can bind to a receptor with minimal steric hindrance of the DNA binding protein.

The term "presentation on the surface of a mammalian cell" refers to a technique whereby an antibody, or a part of an antibody, is expressed and presented on the surface of a mammalian host cell for examination purposes; for example, by examination to determine specific antigen binding by a combination of magnetic beads and fluorescence activated cell sorting. In one aspect, mammalian expression vectors are used for simultaneous expression of immunoglobulins both as a secreted form and a cell surface-bound form as in DuBridge et al., US 2009/0136950. In another aspect, the techniques of Gao et al. for a viral vector encoding an antibody library or antibody fragments they are presented in cell membranes when expressed in a cell as in Gao et al., US 2007/0111260. The surface presentation of complete IgG in mammalian cells is known. For example, Akamatsuu et al. they developed a presentation vector on the surface of a mammalian cell, suitable for directly isolating IgG molecules based on their affinity for antigen binding and biological activity. Using an episomic vector derived from Epstein-Barr virus, antibody libraries were presented as complete IgG molecules on the cell surface and examined to determine specific antigen binding by a combination of magnetic beads and fluorescence-activated cell sorting. Plasmids encoding antibodies with desired binding characteristics were recovered from the classified cells and became the form for the production of soluble IgG. Akamatsuu et al. J. Immunol. Methods 2007 327 (1-2): 40-52. Ho et al. they used 293T human embryonic kidney cells that are widely used for the transient expression of proteins for the presentation on the cell surface of single chain Fv antibodies for affinity maturation. Cells expressing an uncommon mutant antibody with higher affinity were enriched, 240 times by a single pass cell classification from a large excess of cells expressing WT antibody with a slightly lower affinity. In addition, a highly enriched mutant with increased binding affinity for CD22 was obtained after an individual selection from a combinatorial library by randomizing an intrinsic antibody hot spot. Ho et al. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells, Proc Natl Acad Sci U S June 20, 2006; 103 (25): 9637-9642.

Beerli et al. they used specific B cells for an antigen of interest that were isolated directly from peripheral blood mononuclear cells (PBMC) from human donors. Recombinant single chain (scFv) libraries specific for antigen from this combination of B cells are generated and examined by presentation on the surface of a mammalian cell using a Sindbis virus expression system. This method allows to isolate specific antigen antibodies by means of an individual batch of FACS. Variable regions (VR) of heavy chains (HC) and light chains (LC) were isolated from positive clones and recombinant fully human antibodies produced as complete Fab or IgG fragments. In this way, several high-affinity hypermuted antibodies that bind to the pseudoviral particle (VLP) Qb, a model viral antigen, as well as antibodies specific for nicotine were isolated. All antibodies showed high levels of expression in cell culture. Human nicotine-specific mAbs were validated preclinically in a mouse model. Beerli et al., Isolation of human monoclonal antibodies by mammalian cell display, Proc Natl Acad Sci USA. September 23, 2008; 105 (38): 14336-14341.

Also known is the presentation on the cell surface of yeasts, for example, see Kondo and Ueda 2004, Yeast cell-surface display-applications of molecular display, Appl. Microbiol Biotechnol., 64 (1): 28-40, which describes, for example, a system of modification by cell surface engineering using the yeast Saccharomyces cerevisiae. Several representative presentation systems for S. cerevisiae yeast expression are described in Lee et al, 2003, Microbial cell-surface display, TRENDS in Bitechnol. 21 (1): 45-52. Also Boder and Wittrup 1997, Yeast surface display for screening combinatorial polypeptide libraries, Nature Biotechnol., 15 (6): 553.

The term "manufacture" refers to the production of an antibody in an amount sufficient to allow at least clinical phase I trials of a therapeutic antibody, or an amount sufficient for regulatory approval of a diagnostic antibody.

The term "sense change mutation" refers to a point mutation in which an individual nucleotide is changed, which results in a codon encoding a different amino acid. Mutations that change an amino acid to a termination codon are called nonsense mutations.

As used herein, a "molecular property to be evolved" includes the reference to molecules that are composed of a polynucleotide sequence, molecules that are composed of a polypeptide sequence, and molecules that are composed in part of a polynucleotide sequence and in part of a polypeptide sequence. Particularly relevant examples, but not limiting in any way, of molecular properties that are to be evolved include enzymatic activities under conditions

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specified, such as temperature related; salinity; Pressure; pH; and concentration of glycerol, DMSO, detergent, and / or any other molecular species with which it comes into contact in a reaction environment. Additional particularly relevant examples, but not limiting in any way, of molecular properties to be evolved include stabilities, for example, the amount of a residual molecular property that is present after a specified exposure time to a specified environment, such as It can be found during storage.

The term "mutate" refers to creating a mutation in a nucleic acid sequence; in the case where the mutation occurs within the coding region of a gene, which will lead to a codon change that may or may not lead to a change in amino acid.

The term "mutations" means changes in the sequence of a wild-type nucleic acid sequence or changes in the sequence of a peptide or polypeptide. Such mutations can be point mutations such as transitions or transitions. Mutations can be deletions, insertions or duplications.

As used herein, the degenerate "N, N, G / T" nucleotide sequence represents 32 possible triplets, where "N" can be A, C, G or T.

As used herein, the degenerated "N, N, N" nucleotide sequence represents 64 possible triplets, where "N" can be A, C, G or T.

The term "which occurs naturally" as used herein applied to the object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory, occurs naturally. Generally, the term occurs naturally refers to an object present in a non-pathological individual (not sick), such as being typical for the species.

As used herein, a "nucleic acid molecule" is composed of at least one base or a couple of bases, depending on whether it is single stranded or double stranded, respectively. In addition, a nucleic acid molecule may belong exclusively or chimerically to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids , non-genomic nucleic acids, naturally occurring and non-naturally occurring nucleic acids, and synthetic nucleic acids. This includes, by way of non-limiting example, nucleic acids associated with any article, such as mitochondria, ribosomal RNA, and nucleic acid molecules that are chimerically composed of one or more components that do not occur naturally together with components that occur naturally.

Additionally, a "nucleic acid molecule" may in part contain one or more non-nucleotide-based components as exemplified by, but not limited to, amino acids and sugars. Thus, by way of example, but not limitation, a ribozyme that is based in part on nucleotides and based on part in prothene is considered a "nucleic acid molecule."

In addition, by way of example, but not limitation, a nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive label or alternatively a non-radioactive label, is also considered a "nucleic acid molecule."

The terms "nucleic acid sequence encoding for" or a "DNA coding sequence for" or a "nucleotide sequence encoding for" a particular antibody, as! Like other terms synonymous, they refer to a DNA sequence that is transcribed and translated to give an antibody when placed under the control of appropriate regulatory sequences. A "promoter sequence" is a regulatory region of DNA that can bind to RNA polymerase in a cell and initiate the transcription of a subsequent coding sequence (in the sense of 3 '). The promoter is part of the DNA sequence. This sequence region has an initiation codon at its 3 'end. The promoter sequence includes the minimum number of bases when there are the necessary elements to initiate transcription at detectable levels above the background. However, after the RNA polymerase is bound to the sequence and transcription begins at the initiation codon (3 'end with a promoter), the transcription proceeds further in the 3' direction. Within the promoter sequence will be a transcription initiation site (conveniently defined by mapping with the SI nuclease) as! as binding domains to protelna (consensus sequences) responsible for the binding of RNA polymerase.

The terms "nucleic acid encoding an antibody" or "DNA encoding an antibody" or "polynucleotide encoding an antibody" and other synonymous terms encompass a polynucleotide that includes only the coding sequence for the antibody as! as a polynucleotide that includes additional coding and / or non-coding sequence.

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In a preferred embodiment, a "species of specific nucleic acid molecule" is defined by its chemical structure, as exemplified by, but not limited to, its primary sequence. In another preferred embodiment, a specific "nucleic acid molecule" is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species. Therefore, by way of non-limiting example, a "species of specific nucleic acid molecule" can be defined by one or more activities or properties attributable thereto, including activities or properties attributable to its expressed product.

The present definition of "assembling a working nucleic acid sample into a nucleic acid library" includes the method of incorporating a nucleic acid sample into a vector-based collection, such as by linking to a vector and transforming a host. A description of vectors, hosts and other relevant reagents as follows is provided herein. as specific non-limiting examples thereof. The present definition of "assembling a working nucleic acid sample into a nucleic acid library" also includes the method of incorporating a nucleic acid sample into a non-vector-based collection, such as by attachment to adapters. Preferably, the adapters can be paired with PCR primers to facilitate PCR amplification.

Therefore, in a non-limiting embodiment, a "nucleic acid library" is composed of a vector-based collection of one or more nucleic acid molecules. In another preferred embodiment, a "nucleic acid library" is composed of a non-vector collection of nucleic acid molecules. In yet another preferred embodiment, a "nucleic acid library" is composed of a combined collection of nucleic acid molecules that is based in part on vector and not based on part in vector. Preferably, the collection of molecules comprising a library can be subject to search and separation according to the individual nucleic acid molecule species.

The present invention provides a "nucleic acid construct" or alternatively a "nucleotide construct" or alternatively a "DNA construct." The term "construct" is used herein to describe a molecule, such as a polynucleotide (eg, a phytase polynucleotide) that can optionally chemically bind to one or more additional molecular moieties, such as a vector, or parts of a vector In a specific aspect, but not limiting in any way, a nucleotide construct is exemplified by a DNA expression construct suitable for the transformation of a host cell.

An "oligonucleotide" (or synonymously an "oligo") refers to either a single stranded polydeoxynucleotide or two complementary strands of polydeoxynucleotide that can be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5'-phosphate. Those who do not have it will not bind with another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. An oligonucleotidosintetico is ligated with a fragment that has not been dephosphorylated. In order to achieve polymerase-based amplification (such as with PCR), a "32-fold degenerate oligonucleotide that is comprised of, in series, at least a first homologous sequence, a degenerated N, N, G / T sequence, and a degenerated sequence are mentioned. second homologous sequence ”. As used in this context, "homologue" refers to homology between the oligo and the parental polynucleotide that is subjected to polymerase-based amplification.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when placed in a functional relationship placed with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operationally linked means that the DNA sequences that bind are normally contiguous and, when necessary, join two coding regions of protelna, contiguous and in the reading frame.

A coding sequence is "operably linked to" another coding sequence when the RNA polymerase will transcribe the two coding sequences into an individual mRNA, which is then transcribed to give an individual polypeptide having amino acids derived from both coding sequences. It is not necessary for the coding sequences to be contiguous with each other as long as the expressed sequences are ultimately processed to produce the desired antibody.

As used herein, the term "physiological conditions" refers to temperature, pH, ionic strength, viscosity, and similar biochemical parameters that are compatible with a viable organism, and / or that normally exist intracellularly in a cell. of mammalian or viable cultured yeast cell. For example, the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions. In vitro reaction conditions suitable for in vitro transcription cocktails are generally physiological conditions. In general, in vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45 ° C and divalent cation (eg, Mg ++, Ca ++) 0.00110 mM; preferably 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent of non-specific protein (eg, BSA). Often a non-ionic detergent (Tween, NP-40, Triton X-100), usually at 0.001 to 2%, usually 0.05-0.2% (v / v) may be present. They can

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Select particular aqueous conditions by the professional according to conventional methods. For general guidance, the following buffered aqueous conditions may be applied: 10-250 mM NaCl, 550 mM Tris HCl, pH 5-8, with optional addition of divalent cation (s) and / or metal chelants and / or non-detergents ionic and / or membrane fractions and / or antifoaming agents and / or scintillators.

The term "population" as used herein means a collection of components such as polynucleotides, parts of polynucleotides or antibodies. A "mixed population" means a collection of components that belong to the same family of nucleic acids or antibodies (ie, are related) but differ in sequence (ie, are not identical) and so on! in its biological activity.

A molecule that has a "pro-form" refers to a molecule that undergoes any combination of one or more covalent and non-covalent chemical modifications (eg, glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature or pH-induced conformational change, association with a cofactor, etc.) as a way to achieve a more mature molecular form that has a property difference (for example an increase in activity) compared to the reference pro-form molecule. When two or more chemical modifications can be distinguished (for example two proteolytic cleavages, or a proteolytic cleavage and a deglycosylation) as a route for the production of a mature molecule, the reference precursor molecule may be referred to as a "pre-pro-forma" molecule.

A "property" can describe any characteristic, including a physical, chemical or activity characteristic property of an antibody to be optimized. For example, in certain aspects, the property, characteristic or predetermined activity to be optimized can be selected from reduction of antibody-protein aggregation, enhancement of antibody stability, increase in antibody solubility, increase in pH stability of antibody, increase in temperature temperature stability, increase in antibody solvent stability, increase in selectivity, decrease in selectivity, introduction of glycosylation sites, introduction of conjugation sites, reduction of immunogenicity, potentiation of the expression of proteins, increase in antigenic affinity, decrease in antigenic affinity, change in binding affinity, change in immunogenicity, change in catalytic activity, optimization of pH or potentiation of specificity. An "optimized" property refers to a desirable change in a particular property in a mutant antibody compared to a template antibody.

As used herein, the term "pseudorandom" refers to a set of sequences that have limited variability, such that, for example, the degree of residue variability in another position, except any pseudorandom position, is allowed. certain degree of variation of residues, however circumscribed.

"Almost repetition units", as used herein, refers to the repetitions that are to be reordered and are by definition not identical. Indeed, the method is proposed not only for practically identical coding units produced by mutagenesis of the identical starting sequence, but also the reordering of similar or related sequences that can diverge significantly in some regions. However, if the sequences contain enough homologs to be rearranged by this approach, they can be referred to as "almost repeating" units.

As used herein "random peptide library" refers to a set of polynucleotide sequences encoding a set of random peptides, and the set of random peptides encoded by those polynucleotide sequences, as! as to the fusion proteins that contain these random peptides.

As used herein, "random peptide sequence" refers to an amino acid sequence that is composed of two or more amino acid monomers and is constructed by a stochastic or random procedure. A random peptide may include framework motifs or framework region, which may comprise invariant sequences.

As used herein, "receptor" refers to a molecule that has an affinity for a given ligand. The receptors can be molecules that occur naturally or synthetically. The receptors can be used in an unchanged state or as aggregates with other species. The receptors can be covalently or non-covalently bound to a binding member, either directly or through a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as in viruses, cells, or other materials).

"Recombinant" antibodies refer to antibodies produced by recombinant DNA techniques, that is, produced from cells transformed by an exogenous DNA construct encoding the desired antibody. "Synthetic" antibodies are those prepared by chemical synthesis.

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The term "related polynucleotides" means that regions or zones of the polynucleotides are identical and regions or zones of the polynucleotides are heterologous.

"Reductive rearrangement," as used herein, refers to the increase in molecular diversity that accumulates through deletion (and / or insertion) events that are mediated by repeated sequences.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percent sequence identity" and "substantial identity".

A "reference sequence" is a defined sequence used as the basis for a sequence comparison; A reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or genetic sequence given in a sequence list, or it may comprise a complete genetic or cDNA sequence. Generally, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides can each comprise (1) a sequence (that is, a part of the complete polynucleotide sequence) that is similar between the two polynucleotides and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons are usually made between two (or more) polynucleotides by comparing the sequences of the two polynucleotides along a "comparison window" to identify and compare local regions of sequence similarity.

"The Repeat Index (RI)", as used herein, is the average number of copies of the almost repeat units contained in the cloning vector.

The term "saturation" refers to an evolution technique in which every possible change is made in each position of a template polynucleotide or template polypeptide; however, the change in each position is not confirmed by tests, but is merely assumed statistically when it is estimated that most of the possible changes occur or almost every possible change in each position of a mold.

The term "sequence identity" means that two polynucleotide sequences are identical (ie, nucleotide by nucleotide) along the comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences throughout the comparison window, determining the number of positions in which the identical nucleic acid base appears (eg, A, T, C, G, U or I) in both sequences to provide the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e. window size), and multiplying the result per 100 to provide the percent sequence identity. This "substantial identity", as used herein, indicates a characteristic of a polynucleotide sequence, in which the polynucleotide comprises a sequence having a sequence identity of at least 80 percent, preferably an identity of at least 85 percent, often a sequence identity of 90 to 95 percent, and most commonly a sequence identity of at least 99 percent compared to a reference sequence of a comparison window of at least 25-50 nucleotides, in which the percentage of sequence identity is calculated by comparing the reference sequence with the polynucleotide sequence that can include deletions or additions that total 20 percent or less of the reference sequence along from the comparison window.

The term "silent mutation" refers to a codon change that does not result in a change of amino acid in an expressed polypeptide and is based on the redundancy of codon usage for amino acid insertion.

As is known in the art, the "similarity" between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one protein with the sequence of a second protein. Similarity can be determined by procedures that are well known in the art, for example, a BLAST (Basic Local Alignment Search Tool) program at the National Center for Biological Information.

As used herein, the term "single chain antibody" refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked through a spacer peptide (eg, [Gly -Gly-Gly-Gly-Ser] x), and which may comprise additional amino acid sequences at the terminal amino and / or carboxyl ends. For example, a single chain antibody may comprise an anchor segment for binding to the coding polynucleotide. As an example, an scFv is a single chain antibody. Single chain antibodies are generally proteins that consist of one or more polypeptide segments of at least 10 contiguous amino acids encoded substantially by genes of the

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immunoglobulin superfamily (for example, see Williams and Barclay, 1989, pp. 361-368), most frequently encoded by a genetic sequence of heavy chain or rodent light chain, non-human primate, poultry, swine, bovine , sheep, goat or human. A functional single chain antibody generally contains a sufficient part of a gene product from the immunoglobulin superfamily so that the binding property to a specific target molecule, usually a receptor or antigen (epltopo), is retained.

It is said that the members of a pair of molecules (for example, an antibody-antigen pair or a pair of nucleic acids) "specifically bind" to each other if they bind to each other with greater affinity than to other non-specific molecules. For example, an antibody produced against an antigen to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen.

"Specific hybridization" is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (eg, a polynucleotide having a different sequence but substantially identical to the first polynucleotide), in which substantially non-polynucleotide sequences related do not form hybrids in the mixture.

The term "specific polynucleotide" means a polynucleotide that has certain endpoints and that has a certain nucleic acid sequence. Two polynucleotides in which a polynucleotide has the identical sequence as part of the second polynucleotide but different ends comprises two different specific polynucleotides.

"Rigorous hybridization conditions" means that hybridization will occur only if there is an identity of at least 90%, preferably an identity of at least 95% and most preferably an identity of at least 97% between the sequences. See Sambrook et al., 1989.

Also included in the invention are polypeptides that have sequences that are "substantially identical" to the sequence of a polypeptide, such as one of any SEQ ID NO disclosed herein. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence only in substitutions of conservative amino acids, for example, substitutions of one amino acid with another of the same class (eg, substitution of a hydrophobic amino acid, such as isoleucine, valine, leucine or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).

Additionally, a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence in one or more non-conservative substitutions, deletions or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its behavioral properties. For example, one or more amino acids can be deleted from a phytase polypeptide, which results in the modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino or carboxyl terminal amino acids that are not required for the biological activity of phytase can be removed. Such modifications may result in the development of smaller active phytase polypeptides.

As used herein, "substantially pure" means that an object species is the predominant species present (ie, on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably, Substantially purified fraction is a composition in which the target species comprises at least 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than 80 to 90 percent of all macromolecular species present in the composition. Most preferably, the target species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) in which the composition consists essentially of an individual macromolecular species. Solvent species, small molecules (<500 Dalton) and elemental ionic species are not considered macromolecular species.

As used herein, "template oligopeptide" means a template antibody for which a secondary library of variants is desired. As those skilled in the art will appreciate, any number of molds find use in the present invention. Specifically within the definition of "antibodies" or "oligopeptides" fragments and known antibody domains are included, including functional domains such as binding domains, etc., and smaller fragments, such as spins, loops, etc. That is, parts of an antibody can also be used. In addition, antibody variants, that is, structures analogous to antibodies that do not occur naturally, can be used.

Suitable principal structures of an antibody include, but are not limited to, all those found in the database compiled and maintained by the Research Collaborative for Structural Bioinformatics (RCSB, formerly the Brookhaven National Laboratory).

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As used herein, the term "variable segment" refers to a part of a nascent peptide comprising a random, pseudorandom or defined nucleus sequence. A "variable segment" refers to a part of a nascent peptide comprising a random, pseudorandom or defined nucleus sequence. A variable segment may comprise both variant and invariant residue positions, and the degree of variation of residues in a variant residue position may be limited: both options are selected at the discretion of the professional. Typically, the variable segments are 5 to 20 amino acid residues in length (for example, 8 to 10), although the variable segments may be longer and may comprise antibody parts, such as an antibody fragment.

The term "wild" or "wild type" means that the polynucleotide does not comprise any mutation. A "wild type" antibody means that the antibody will be active at a level of activity that is found in nature and will comprise the amino acid sequence found in nature.

Detailed description of the invention

The disclosure provides a method of integration of generation and / or selection, evolution and expression of therapeutic antibodies in a mammalian host for manufacturing in an individual system. In an embodiment of the CIAO method, therapeutic antibodies are generated, optimized and manufactured in the same mammalian host system. In another embodiment, protein therapeutic agents are discovered and manufactured in the same host. This optimizes manufacturing from the very beginning, saving costs (time and resources).

Historically, antibody discovery has been performed in eukaryotic (euc) and prokaryotic (proc) hosts. Normally, in bacteria (E. coli), partial length antibodies are discovered; for example, in phage presentation technologies, Fab is recovered and sometimes they become full length later. There are several possible disadvantages in these approaches.

In one example, there is some evidence that regions of Fc and Fv communicate to affect antibody properties, such as binding and expression. Therefore, when an antibody fragment is optimized for a property such as expression, the improvement does not always translate into an improved expression in the full length assembled antibody. For example, an Fc library was created as attempts to find a "holy grail" Fc that could join any Fv to improve expression on any guest.

In one aspect, codon mutagenesis was performed in the constant region for the optimization of mammalian cell expression. Specifically, 326 mutants were created in the constant region and expressed in HEK 293 and CHO-S cells. An ELISA exam was performed. Several Fc meet the criteria for enhanced expression, and even certain optimized Fc that transfer positive effects across multiple cell lines were identified; however, when a different Fv was attached to the Fc, the improvement did not translate into expression. This shows that those that Fc and Fv communicate.

To avoid unexpected results after recombination of antibody fragments, in a preferred aspect, the CIAO method is used to discover full-length antibody molecules. In another preferred aspect, the CIAO method uses euc guests.

In one embodiment, the eukaryotic system is a mammalian system that is selected from one of the group consisting of the CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549 cell lines , A375, SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT- 474, Caco- 2, HCC 1954, MDAMB-468, LnCAP, NRK-49F and SP2 / 0; and mouse splenocytes and rabbit PBMC. In one aspect, the mammalian system is selected from a CHO or HEK293 cell line. In a specific aspect, the mammalian system is a CHO-S cell line. In another specific aspect, the mammalian system is a HEK293 cell line. In another embodiment, the eukaryotic system is a system of yeast cells. In one aspect, the eukaryotic system is selected from S. cerevisiae yeast cells or Picchia yeast cells.

In another embodiment, the creation of mammalian cell lines can be done commercially by a contract research organization or custom manufacturing. For example, for recombinant or other protein antibodies, Lonza (Lonza Group Ltd, Basel, Switzerland) can create vectors to express product using the GS Gene Expression System ™ technology with the host cell lines either CHOK1SV or NSO.

In another embodiment, evolution may be performed on proc guests (such as E. coli) and examinations may be performed on euc guests (eg, CHO), provided that the examination occurs on the same host as the manufacturing guest.

Selection of candidate antibody (s) llder

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A variety of methods can be used to discover, generate and / or select one or more candidates for therapeutic antibody to be evolved. The selected molecules may be existing recombinant antibodies, or they may come from collections of recombinant antibodies. Recombinant antibodies can be discovered using any number of available generation and test platforms. The antibodies can be in any form, including single chain, fully human, Fab, Fv, Fc, bifunctional, chimeric, humanized or fully human antibodies or fragments thereof. In a preferred aspect, the CIAO method is used to discover full-length antibody molecules. Recombinant antibody libraries can be generated and examined using screening or screening systems in mammalian hosts optimized or not optimized to provide candidates for evolution. Several eukaryotic expression systems are published, for example, mammalian or yeast cell systems.

In the method of the present invention, such mammalian expression systems, in particular systems that use presentation on the cell surface of molecules for examination and selection, are used to identify and select candidates for fabrication, or evolution followed by fabrication. Preferably, such mammalian hosts are fibroblast cells (3T3, mouse; BHK21, Syrian hamster), epithelial cells (MDCK, dog; Hela, human being; PtK1, rat, kangaroo), plasma cells ((SP2 / 0 and NS0, mouse ), kidney cells (293, human; COS, mono), ovarian cells (CHO, Chinese hamster), embryonic cells (R1 and E14.1, mouse; H1 and H9, human; PER C.6, be human.) The presentation technology on the cell surface is used to present antibodies on the surface of the mammalian cells for examination. Proteins are cloned as fusions with membrane molecules that, when expressed, present the proteins on the surface of the cells. for a high performance exam, fast, for example.

In certain embodiments, the disclosure provides a method of provision of an optimized antibody. In one embodiment, an antigen is selected and a library of human antibodies is generated and expressed in a mammalian system, for example, CHO-S cells. The library is examined to identify fully human antibody matches, for example, by fluorescence activated cell sorting (FACS). Fully human antibody matches are then examined / further characterized by any relevant assay to determine at least one predetermined property, characteristic or activity. In one aspect, evolved molecules are examined to determine multiple characteristics simultaneously, for example, improved function and expression. The relevant assay may comprise, for example, ELISA, or an alignment technology. A template antibody is selected from the human antibody matches. Any method of evolution is performed with the template antibody, or a fragment polypeptide thereof, to prepare a set of mutant antibodies. In one aspect, the evolution is carried out by means of a modification method by means of complete protein engineering to produce the set of mutant antibodies. The method of modification by full-length engineering can be selected, for example, from one or a combination of complete position evolution (CPE ™), complete protein synthesis (CPS ™), flexible evolution, synergistic evolution, position insertion evolution complete (CPI ™) or complete position deletion evolution (CPD ™). In another aspect, the mutant antibodies are expressed in the same mammalian system used to generate the human antibody library.

The set of mutant antibodies are characterized / examined to determine at least one predetermined property, characteristic or activity. In one aspect, the set of mutant antibodies is examined simultaneously, for example, to determine improved function and expression. In one aspect, a specific molecule database is used in the form of a functional position map (an EvoMap ™) for further optimization by one or more protein evolution techniques known in the art; followed by identification and characterization of higher mutants. An optimized antibody is selected by comparison with the template antibody with respect to the at least one predetermined property, characteristic or activity.

In one aspect, gram-scale expression of the selected optimized antibody is performed; followed by toxicology other than LPG. The stable cell line transfection, the procedure development and the generation of a master cell bank are performed. Manufacturing with BPF is performed in the same mammalian system used to generate the human antibody library, for example, CHO-S cells, to result in Acm optimized therapeutic recombinants.

In another embodiment, the CIAO method starts from the selection of a hybridoma template or recombinant antibody; the evolution of the antibody to provide a set of mutant antibodies that are examined by the use of antibody cell surface presentation in a mammalian cell system; and manufacturing is done in the same mammalian cell system used for the exam.

In another embodiment, the selected template hybridoma / recombinant antibody is humanized and examined in the manufacturing host, followed by production in the manufacturing host, in which the optimization (evolution) stage is completely omitted.

In other aspects of the present invention, the optimization of subsequent expression in manufacturing hosts is carried out by evolving the Fc region of the antibody, silent codons in the antibody, and / or the vector and / or

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host genes used in the expression of proteins. In one aspect, an Fc library is generated by any evolutionary technique. In a specific aspect of expression optimization, CPE is performed with an Fc domain of an antibody to create a library of Fc mutants that can be used to select an optimal partner for any Fv. The optimization is designed for a quick union of all CPE variants of Fc to each new Fv region. Alternatively, a subset of these Fc can be used to join different Fv. Each of these combinations of Fc / Fv CPE variant is examined as a full-length antibody expressed in mammalian cells (eg CHO, cost-effective means) for optimal expression. In addition, CPS can be performed to examine all theoretical permutations of up to 12 or more of these CPE matches in mammalian cells to determine improved expression. Specific desirable codon changes can also be selected to identify clones with increased expression. Silent codons are identified and cPe is performed in these positions. This CPE library is examined to identify optimal expression matches. In addition, all theoretical permutations of up to 12 or more CPE matches can be used in the CPS procedure to generate a new library that can be examined in mammalian cells to determine an improvement in expression. The superior CPS silent mutation matches are used to customize the protein for optimal expression in a cell line and specific media. This provides the opportunity to control the fine structure of biosimilar drugs.

Other areas for potentiating expression include: optimization of the vector, including promoter, splice sites, 5 'and 3' terminal ends, flanking sequences, reduction of deletion and genetic transposition, improvement of host cell gene activities , optimization of host glycosylation enzymes, and mutagenesis and selection of host cells of chromosomal width. It has been shown that 5 'amino acid sequences are important for potentiating expression.

Evolution of candidates llder

Any method of evolution of proteins can be used for the simultaneous evolution of performance optimization and antibody expression. Optimization of antibody performance may include the improvement of various characteristics such as affinity, pharmacokinetic characteristics, tissue targeting, antibody-protein aggregation, direction of high assay variability and modification of other characteristics in vivo.

Methods for evolving molecules, including selected candidates of the present invention, include stochastic and non-stochastic methods. Published methods include approaches to random and non-random mutagenesis. Any of these approaches can be used to evolve properties of the therapeutic antibodies of the present invention towards a desired characteristic, such as better stability at different pH or temperature environments, or better expression in a host cell. Other potentially desirable properties, such as improved catalytic activity, improved protein stability under various conditions, improved selectivity and / or solubility, and improved expression results by improving features such as reduced aggregation can be selected for evolutionary experiments.

The evolution is performed directly on a eukaryotic host, such as a mammalian cell host or a yeast cell host, which will be used for the subsequent production of the therapeutic antibody. Candidates can be evolved for optimal expression in the same host used to examine and / or evolve and for manufacturing. Expression optimization can be achieved by optimizing used vectors (vector components, such as promoters, splicing sites, 5 'and 3' terminal ends and flanking sequences), genetic modification of host cells to reduce deletions and transpositions genetics, evolution of host cell gene activities through in vivo or in vitro methods of evolving relevant genes, optimization of host glycosylation enzymes through the evolution of relevant genes, and / or through mutagenesis strategies and selection of host cells wide Chromosome to select for cells with enhanced expression capabilities. Guest cells are further described herein.

The examination and expression technology with presentation on the cell surface (for example, as defined above) can be used to examine libraries of evolved antibodies to determine candidates to be manufactured.

Current methods of wide use to create mutants from a starting molecule are oligonucleotide-directed mutagenesis technologies, error-prone polymerase chain reactions and cassette mutagenesis, in which the specific region to be optimized is replaced. by a synthesized mutagen oligonucleotide. In these cases, several mutant sites are generated around certain sites in the original sequence.

In oligonucleotide-directed mutagenesis, a short sequence is replaced by a synthesized mutagen oligonucleotide. Error-prone PCR uses low fidelity polymerization conditions to introduce a low level of point mutations randomly along a long sequence. In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the

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mixture. In cassette mutagenesis, a block of sequences from an individual template is usually replaced by a (partially) randomized sequence.

Chimeric genes have been produced by joining 2 polynucleotide fragments using compatible cohesive ends generated by restriction enzyme (s), in which each fragment is derived from an independent parent (or parental) molecule. Another example is the mutagenesis of an individual codon position (ie to achieve a substitution, addition or deletion of codon) in a parental polynucleotide to generate an individual progeny polynucleotide encoding a mutagenized polypeptide at the individual site.

In addition, site-specific recombination systems have been used in vivo to generate gene hybrids, as well! as random methods of recombination in vivo, and recombination between homologous but truncated genes in a plasmid. Mutagenesis has also been reported by overlapping extension and PCR.

Non-random methods have been used to achieve greater numbers of point mutations and / or chimerizations, for example complete or exhaustive approaches have been used to generate all molecular species within a particular cluster of mutations, to attribute functionality to specific structural groups in a template antibody (for example a specific individual amino acid position or a sequence consisting of two or more amino acid positions), and for classifying and comparing specific groups of mutations. US Patent No. 7033781 entitled "Whole cell engineering by mutagenizing a sustantial portion of a starting genome, combining mutations, and optionally repeating" describes a method of evolving an organism toward desired characteristics. US patent number 6764835 entitled "Saturation mutagenesis in directed evolution" and US patent number 6562594 entitled "Synthetic linkage reassembly in directed evolution" describes methods of evolving exhaustively and examining to determine desired molecule characteristics. Any such method can be used in the method of the present invention.

There is a difference between the previously known methods of "saturation mutagenesis" and the "complete" evolution techniques preferred herein. Saturation mutagenesis refers to an evolution technique in which every possible change is made in each position of a template polynucleotide or template polypeptide; however, the change in each position is not confirmed by tests, but is merely assumed statistically. Complete evolution refers to an evolution technique in which every possible change is made in each position of a template polynucleotide or template polypeptide and the polynucleotide or polypeptide is tested to confirm that the intended change has been made.

The saturation methods are inherently statistical methods, not complete and were never truly complete through all stages (for example, through mutant generation, mutant identification, mutant protein expression, mutant protein screening and generation, identification, expression and examination of recombinant higher mutants). In complete evolution techniques, each molecule is examined and confirmed both in the first stage of mutagenesis, as well as in a second stage of recombination of the upper mutants or coincidences.

Unless saturation mutagenesis is confirmed by sequencing or some other method, the technique cannot be considered complete for several possible reasons. For example, 1) the cloning systems are not 100% effective due to cloning or synthesis errors, or it is difficult to clone the molecules or 2) some proteins are toxic when expressed and therefore cannot be expressed effectively. Therefore, it is important to confirm by sequencing, or some other technique, at each stage. It is useful to rate each stage to examine the expression, so that clones without expression are not designated as "negative" as in the previous work, they are only scored as non-expressible. Therefore, complete techniques are considered to be a purer non-stochastic system than saturation techniques, as confirmed by the "confirmation" stage.

Full Position Evolution

Referring to Figure 1, using a linear peptide as a simple example, in a first stage, a set of naturally occurring amino acid variants (or a subset thereof, or amino acid derivatives) is generated for each codon. from position 1 to n (corresponding n to the number of residues in the polypeptide chain) by a procedure referred to herein as complete position evolution (CPE ™). This procedure is repeated for each polypeptide chain of the target molecule. A minimum set of amino acid mutations contains only one codon for each of the 19 natural amino acids. However, it is recognized that each expression system may experience codon bias, in which insufficient combinations of tRNAs can lead to translation stoppage, premature translation termination, translation frame shift and incorrect amino acid incorporation. Therefore, for the optimization of the expression, each set contains up to 63 different codons, including termination codons. In the next stage, the mutations are confirmed by sequencing each new molecule. Other confirmation methods can also be used.

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Each set of amino acids is then examined to determine at least one of:

- Enhanced function

- Neutral mutations

- Inhibitory mutations

- Expression

- Compatibility of the clone with the host system.

Preferably, multiple features are examined simultaneously such as, for example, improved function and expression.

The data for each set is combined for the entire polypeptide chain (s) and a detailed functional map (referred to herein as EvoMap ™) of the target molecule is generated. This map contains detailed information on how each mutation affects the performance / expression and / or cloning capacity of the target molecule. It allows the identification of all sites where changes cannot be made without a loss of function of the protein (for example, antigen / receptor binding in case of antibodies). It also shows where changes can be made without affecting the function. It also identifies changes that result in molecules that are not expressed in the host system, and therefore do not evaluate the effect of the mutation.

In Figure 1, a schematic of a hypothetical EvoMap ™ is shown. Each position in the mold is identified as a restricted (non-mutable) site, a totally mutable site, a partially mutable site or a higher mutant for a specific amino acid substitution. Each partially mutable site may also be designated as suitable for substitution, for example, by a charged residue, or a substitution of apolar residue, and a clone without expression and / or molecule that cannot be cloned into the host system.

It is possible to use the EvoMap ™ to recognize and recombine beneficial individual amino acid substitutions, and examine to further optimize the desired characteristics in the target molecule. However, the evolution of certain characteristics may require two or more simultaneous mutations to be observed. The EvoMap ™ can be used to efficiently and profitably produce a set of multi-site mutant polypeptides non-randomly. The set of multi-site mutant polypeptides can then be examined to determine higher multi-site mutants.

The CPE allows the map of antibody mutations confirmed in complete vivo. The identification of the whole set of higher mutants allows additional combinatorial evolution stage (s). CPE can be used to reduce the risk of immunogenicity of antibodies evolved by the selection of non-surface mutations; elimination of epltopos from T cells; and imitation of somatic mutations.

In one aspect, CPE can be used to generate a library of up to 5, 10 or 15 amino acids, or up to all 19 amino acids. Changes are made to each position in the antibody and examined to determine a desirable characteristic, such as binding affinity or expression, and Evomap ™ is created. Subsequent batches of mutation and examination can be used to generate the data for the 19 amino acids. From the map, totally mutable sites are identified. These sites are useful for identifying positions that can be modified to create a new collection of molecules that can be produced and tested for characteristics. For example, informatics can be used to identify HLA haplotypes in the sequence, and desired changes can be made to avoid these haplotypes by making specific targeted changes at "neutral" ("fully mutable") sites identified from the map, in which the characteristic Neutral will not be affected. This could potentially reduce the risk of immunogenicity (non-surface mutations could be selected, eliminate T-cell epltopos, mimic hypersomatic mutations). In addition, the map can show sites for specific site modifications (glycosylation and chemical conjugation) to improve various characteristics. In addition, optimization of silent mutations can improve antibody expression in a variety of hosts.

Synergistic evolution

In one embodiment of the present invention, an EvoMap ™ is generated and used for synergistic evolution, as shown in Figure 2. In synergistic evolution, simultaneous mutation can be combined in 2-20 selected sites to produce a combinatorial effect. . The EvoMap ™ of the template polypeptide is used to select individual specific amino acid point mutations for assembly in multi-site polypeptide mutations.

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In synergistic evolution, specific non-deactivating amino acid mutations are selected from within partially mutable sites that are near non-mutable sites in the EvoMap ™. In one aspect, the selected non-deactivating point mutations are adjacent to non-mutable sites. In synergistic evolution, simultaneous amino acid mutation is performed in two to 20 of the sites selected for combinatorial effects. In one aspect, recombination of two to 20 selected mutations is used to produce a codon variant library that encodes a population of multi-site mutant polypeptides. After cloning and expression, the multi-site mutant polypeptides produced are then examined to determine at least one predetermined property, characteristic or activity compared to the template polypeptide. In this way, multi-site higher mutant polypeptides can be identified. In one aspect, multi-site mutant polypeptides are produced by combinatorial synthesis of proteins. An advantage of synergistic evolution is that it does not require an X-ray crystalline structure of the antibody to direct the evolution of the template polypeptide. This technique is particularly useful for antibodies with high assay variation and other multi-site effects.

According to the present invention, the applications of synergistic evolution include, but are not limited to evolution of complex molecular mechanistic changes, evolution of antibodies with high assay variation, evolution of antibody specificity, improvement of expression in various expression hosts. , stability and optimization of pH. The synergistic evolution is applicable to therapeutic antibodies.

In one aspect of the present invention, synergistic evolution can be used to optimize one or more aspects of a polypeptide that is part of an antibody molecule. The antibody molecule can be assembled by binding one or more mutant nucleic acids encoding zero polypeptides, one or more nucleic acids encoding framework region polypeptides to create a variant antibody by cloning, translation and expression techniques known in the art. . In one aspect, a framework region polypeptide is derived from a wild-type antibody molecule. In this aspect, synergistic evolution can be used in conjunction with antibody humanization techniques. For example, a mouse monoclonal antibody can be selected for evolution and humanization. The CDR regions of the antibody are cloned and sequenced and individual CDR regions (CDR1, CDR2, CDR3) can be synthesized and ligated to other nucleotides encoding human antibody framework region polypeptides, followed by the production of a variant IgG library human The human variant IgG library is then examined to determine at least one property, for example two or more properties including improved function and expression, as compared to the mouse Acm. In another aspect, a framework region polypeptide is an artificial framework polypeptide. In the examples section, specific techniques of preparation of bc DNA fragments, ligation and assembly of nucleic acids, cloning, transfection, expression, solid phase synthesis of libraries, synthesis phase of libraries, complete position evolution are presented , combinatorial synthesis of proteins, quantification of expression by quantification by ELISA and b-galactosidase assay and functional ELISA.

In another embodiment of the invention, synergistic evolution can be used to enhance the binding affinity of an antibody. In this embodiment, optimization of the variable region of the antibody can be performed. For example, for the production of antibody mutants, CPE is performed for variable light chain and heavy chain regions of a selected antibody and an EvoMap ™ is generated. Mutants are selected for reassembly; for example, variants of the light chain are selected and variants of the heavy chain are selected for assembly. Point non-deactivating amino acid mutations are selected from within partially mutable sites that are near non-mutable sites. Reassembly technology that CPS uses to create a heavy chain library can be used. Light chain variants can be combined with heavy chain variants, cloned, expressed and variants are examined as complete IgG of eukaryotic cell line supernatants. Binding affinity for certain variants is evaluated by, for example, the use of ELISA, BIAcore and / or Sapidyne instrumentation tests, or other techniques known to one skilled in the art.

Flexible evolution

In another embodiment, CPE / EvoMap can be used to identify and leverage fully mutable sites. In one aspect, the use of multiple fully mutable sites is called flexible evolution and is used to make targeted changes such as introduction of glycosylation sites (eg codons for amino acids for glycosylation linked to N or O; Asn within the Asn consensus sequence -Aa-Ser-Thr or Ser / Thr) and chemical conjugation. Flexible evolution can also be used in the design of cleavage sites with protease, introduction of labels for purification and / or detection, site specific labeling, and the like. In addition, codon optimization of silent mutations can be used to improve antibody expression. In this embodiment, called flexible evolution, after antibody expression, the libraries of mutant polypeptides produced are reexamined to determine at least one predetermined property, characteristic or activity compared to the template polypeptide. In one aspect, the predetermined property includes reduction of antibody-protein aggregation, enhancement of antibody stability, or increase of antibody solubility. In one aspect, libraries of mutant polypeptides are examined to determine two or more properties simultaneously. In another aspect, any eukaryotic expression system that glycosyl can be used for the introduction of glycosylation sites, such as, for example, mammalian cell lines, plants, yeasts and insects.

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In flexible evolution, the evaluation of the bioinformatics and crystalline x-ray structures of antibody related antibodies, or the antibody or template polypeptide, is useful for the optimization of molds. In one aspect, selected sites are not contact residues. In another aspect, the selection of non-surface antibody mutations allows a reduced risk of immunogenicity.

Applications of flexible evolution include, but are not limited to, reduction of antibody-protein aggregation, improvement of antibody solubility, optimization of pharmacokinetics through glycosylation libraries, optimization of secondary and tertiary structure of antibodies and deinmunization of antigenic sites directly through either mutation sets or indirectly through glycosylation masking.

In one aspect of the flexible evolution, an EvoMap ™ is used to identify totally mutable sites, CPS generation is performed with insertion of glycosylation residues in totally mutable sites (or silent mutations for translation purposes), and the examination of the combinatorial glycosylated library by analytical analysis (for example analysis by mass spectrometry, dynamic light scattering), reduction of immunogenicity (by bioinformatics or assay) and / or pharmacokinetic analysis (for example in Foxnlnu mice).

In one aspect, flexible evolution can be used for deimmunization to eliminate immunogenicity while maintaining function. Deimmunization can be performed by flexible evolution masking glycosylation immunogenicity, identification of amino acid substitutions of human hypersomatic mutation spectra that can eliminate immunogenicity while maintaining function, dose reduction to avoid possible immunogenicity, and minimization of changes in amino acid residue not on surface. In addition, immunogenicity databases and algorithms can be used to identify and replace possible CMH binding epltopos. In one aspect, the prediction of modification in silico is coupled with CPE / CPS data to generate variants.

The reduced propensity to generate T-cell epltopos and / or de-immunization can be measured by techniques known in the art. Preferably, the de-immunization of proteins can be tested in vitro by a T cell proliferation assay. In this assay, PBMCs from donors representing> 80% of HLA-DR alleles in the world are examined to determine proliferation in response to polypeptides either natural or deimmunized. Ideally, cell proliferation is only detected after loading antigen presenting cells with wild-type peptides. Additional assays to determine deimmunization include in vitro restimulation assays of human PBMCs (for example ELISA for interferon-gamma (TH1) or IL4 (TH2)). Alternatively, deimmunization can be tested by expressing HLA-DR tetramers representing all haplotypes. To test whether deimmunized peptides are present in HLA-DR haplotypes, the binding of, for example, fluorescently labeled peptides in PBMC can be measured. The measurement of transgenic mice for HLA class I and class II to determine the responses to the target antigen (for example interferon-gamma or IL4). Alternatively, the epltopos library examination with educated T cells (nonameric CMHI; icosameric CMHII) of PBMC and / or transgenic mouse assays. In addition, deimmunization can be tested by determining whether antibodies have been generated against deimmunized molecules after administration in patients.

In another embodiment, the flexible evolution techniques of the present invention can be used for the optimization of expression. In one aspect, the present invention discloses the use of methods of modification by engineering of proteins to develop variants of Fc with codons optimized with silent mutation with improved expression in eukaryotic cells. A silent mutation is one in which a variation of the DNA sequence does not result in a change in the amino acid sequence of the antibody. In one aspect, codon mutagenesis is performed in the constant region for optimization of eukaryotic cell expression. A variant of Fc with optimized codons with improved expression properties while retaining the ability to mediate effector functions improves the production of therapeutic antibodies. In this aspect, for example, a constant region of an antibody molecule can be evolved to examine in different expression hosts, for example, examination of mammalian cell line expression using CHO, HEK293 and COS-7. In Figure 3, an example of optimization of expression by codon mutagenesis in the constant region for mammalian cell expression is shown and described in example 19. The expression levels shown are each an average of 4 points. of data, and were confirmed throughout multiple experiments. The ability of multiple cell lines for the first mutant tested in HEK293 and CHO cell line expression systems was demonstrated.

In addition, EvoMap ™ can be used to generate three-dimensional computational molecular models of the oligopeptide, or specific regions thereof, to explore the structural mechanisms involved in, for example, antibody-epltopo stability and specificity. A hypothetical three-dimensional EvoMap ™ is shown in Figure 13.

The information in EvoMap can also be combined with structural information (if available) to

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select for example, only surface residues for mutations to increase solubility / decrease aggregation.

Evolution of full position insertion

In one embodiment, the disclosure provides methods of identifying and mapping mutant polypeptides formed from, or based on, a template polypeptide. Referring to Figure 8, using a linear peptide as a simple example, in a first stage, a set of naturally occurring amino acid variants (or a subset thereof, or amino acid derivatives) is generated for each codon. from position 2 to n (corresponding n to the number of residues in the polypeptide chain) by a procedure referred to herein as the evolution of full position insertion (CPI ™).

In the CPI ™, an amino acid is inserted after each amino acid in the entirety of a template polypeptide one at a time to generate a set of elongated polypeptides. CPI can be used to insert 1, 2, 3, 4, or up to 5 new sites at a time. Each of the 20 amino acids is added to each new position, one at a time, creating a set of 20 different molecules in each new position added to the mold. In this case, position 1 is skipped, which is methionine and invariant. This procedure is repeated for each polypeptide chain of the target molecule. A minimum set of amino acid mutations contains only one codon for each of the 20 natural amino acids.

The present invention relates to methods of identifying and mapping mutant polypeptides formed from, or based on, a template polypeptide. Normally, the polypeptide will comprise n amino acid residues, in which the method comprises (a) generating n + [20 x (n-1)] independent polypeptides, in which each polypeptide differs from the template polypeptide in which it has been inserted after each position in the mold each of the 20 amino acids one at a time (as illustrated in Figure 1); test each polypeptide to determine at least one predetermined property, characteristic or activity; and (b) for each member to identify any change in said property, characteristic or activity in relation to the template polypeptide.

In one embodiment, one or more regions for mutagenesis are selected to add one position at a time as described above. In that case, n represents a subset or region of the template polypeptide. For example, when the polypeptide is an antibody, the entire antibody or one or more complementarity determining regions (CDR) of the antibody are subjected to mutagenesis to add one position at a time in the template polypeptide after each position.

The invention thus includes mapping methods of a set of mutant antibodies formed from a template antibody having at least one, and preferably six, complementarity determining regions (CDRs), the CDRs together comprising n amino acid residues, the method (a) comprising generating n + [20 x (n-1)] independent antibodies, in which each antibody differs from the template antibody in which an individual predetermined position has been inserted, one at a time, after each position in the sequence mold; (b) test each set to determine at least one predetermined property, characteristic or activity; and (c) for each member to identify any change in a property, characteristic or activity in relation to the template polypeptide. For antibodies, the property, characteristic or predetermined property may be binding affinity and / or immunogenicity, for example.

In addition, production methods of a set of mutant antibodies formed from a template antibody having at least one complementarity determining region (CDR) are provided, the CDR comprising n amino acid residues, the method comprising: (a) generating n + [20 x (n-1)] independent antibodies, in which each antibody differs from the template antibody in that it has an extra amino acid added in a predetermined individual position of the CDR. In another embodiment, the antibody comprises six CDRs, and together the CDRs comprise n amino acid residues.

In another embodiment, the new elongated polypeptides described above are further mutated and mapped after examination to identify a change in a property, characteristic or activity in relation to the shortened polypeptide. Normally, the elongated polypeptide will comprise n amino acid residues, in which the method comprises (a) generating n (n-1 in the case where the initial residue is methionine) independent sets of polypeptides, each set comprising member polypeptides having a number X of predetermined different amino acid residues in a predetermined individual position of the polypeptide; in which each set of polypeptides differs in the predetermined individual position; test each set to determine at least one predetermined property, characteristic or activity; (b) for each member to identify any change in said property, characteristic or activity in relation to the template polypeptide; and optionally (c) create a functional map that reflects such changes. Preferably, the number of different member polypeptides generated is equivalent to n x X (or [n-1] x X, as the case may be).

As an alternative, the method comprises generating an individual population comprising the sets of

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mutated polypeptides of elongated polypeptides. In this embodiment, the entire new population is examined, individual members are identified and the functional map is generated.

Normally, when each naturally occurring amino acid is used, X will be 19 (representing the 20 naturally occurring amino acid residues and excluding the particular residue present in a given position of the template polypeptide). However, any subset of amino acids can be used in the entirety, and each set of polypeptides can be substituted by all or a subset of the total X used for the entire population.

However, it is recognized that each expression system may experience codon bias, in which insufficient combinations of tRNAs can lead to translation stoppage, premature translation termination, translation frame shift and incorrect amino acid incorporation. Therefore, for the optimization of the expression, each set contains up to 63 different codons.

Each set of amino acids is then examined to determine at least one, and preferably two or more, desirable characteristics such as improved function; neutral mutations, inhibitory mutations and expression.

In one aspect, elongated polypeptides can be mapped to identify a change in a property, characteristic or activity that results in shortened polypeptides in relation to the "natural type." The data for each set is combined for the entire polypeptide, or "target molecule." The coincidence of the elongated polypeptide (target molecule) test can then be used for additional complete chain (s) mutagenesis and the test as described herein. The mutagenesis data provides that a detailed functional map (referred to herein as EvoMap ™) of the target molecule is generated. This map contains detailed information on how each mutation affects the performance / expression of the target molecule. It allows the identification of all sites where changes cannot be made without loss of antibody function (or antigen / antibody receptor binding). It also shows where changes can be made without affecting the function.

In another aspect, CPE can be used to generate a library of 5, 10, up to 15, or up to 19 amino acids in each position of interest.

Evolution of full position deletion

The evolution of complete position deletion (CPD ™) refers to methods of identifying and mapping mutant polypeptides formed from, or based on, a template polypeptide. The evolution of CPD deletes each amino acid through the antibody one position at a time. Typically, the polypeptide will comprise n amino acid residues, in which the method comprises (a) generating n-1 (n-2 in the case where the initial residue is methionine) independent polypeptides, in which each polypeptide differs from the polypeptide mold in which it lacks an individual predetermined position; test each polypeptide to determine at least one predetermined property, characteristic or activity; and (b) for each member to identify any change in said property, characteristic or activity in relation to the template polypeptide.

In one embodiment of CPD evolution, one or more regions for mutagenesis are selected to eliminate one position at a time. In that case, n represents a subset or region of the template polypeptide. For example, when the polypeptide is an antibody, the entire antibody or one or more complementarity determining regions (CDR) of the antibody are subjected to mutagenesis to eliminate one position at a time in the template polypeptide.

In one embodiment, CPD therefore includes methods of mapping a set of mutant antibodies formed from a template antibody having at least one, and preferably six, complementarity determining regions (CDRs), the CDRs together comprising n residues. of amino acid, the method comprising (a) generating (n-1) independent antibodies, in which each antibody differs from the template antibody in that it lacks an individual predetermined position; (b) test each set to determine at least one predetermined property, characteristic or activity; and (c) for each member to identify any change in a property, characteristic or activity in relation to the template polypeptide. For antibodies, the predetermined property, characteristic or property may be binding affinity and / or immunogenicity, for example.

One aspect of CPD evolution includes methods of producing a set of mutant antibodies formed from a template antibody that has at least one complementarity determining region (CDR), the CDR comprising amino acid residues, the method comprising: (a) generate n-1 independent antibodies, in which each antibody differs from the template antibody in that it lacks an individual predetermined position of the CDR. In another embodiment, the antibody comprises six CDRs, and together the CDRs comprise n amino acid residues.

In another embodiment of the evolution of CPD, the new shortened polypeptides described above are mutated and

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additionally map after the test to identify a change in a property, characteristic or activity in relation to the shortened polypeptide. Normally, the shortened polypeptide will comprise n amino acid residues, in which the method comprises (a) generating n (n-1 in the case where the initial residue is methionine) independent sets of polypeptides, each set comprising member polypeptides having a number X of predetermined different amino acid residues in a predetermined individual position of the polypeptide; in which each set of polypeptides differs in the predetermined individual position; test each set to determine at least one predetermined property, characteristic or activity; (b) for each member to identify any change in said property, characteristic or activity in relation to the template polypeptide; and (c) create a functional map that reflects such changes. Preferably, the number of different member polypeptides generated is equivalent to n x X (or [n-1] x X, as may be the case).

As an alternative, the CPD method comprises generating an individual population comprising the sets of mutated polypeptides from the shortened polypeptides. In this realization, the entire new population is examined, individual members are identified and the functional map is generated. Normally, when each naturally occurring amino acid is used, X will be 19 (representing the 20 naturally occurring amino acid residues and excluding the particular residue present in a given position of the template polypeptide). However, any subset of amino acids can be used in the totality, and each set of polypeptides can be substituted by all or a subset of the total X used for the entire population.

Any mutation or synthetic means can be used to generate the set of mutants in the evolution of CPD. In one embodiment, the polypeptide generation comprises (i) subjecting a polynucleotide containing codons encoding the polymerase-based template to a polymerase amplification using a degenerate oligonucleotide 64 times for each codon to be mutagenized, in which each of the Degenerated oligonucleotides 64 times is composed of a first homologous sequence and a degenerate N, N, N triplet sequence, so that a set of progeny polynucleotides is generated; and (ii) subjecting the set of progeny polynucleotides to clonal amplification so that polypeptides encoded by the progeny polynucleotides are expressed.

In one embodiment of CPD evolution, all shortened polypeptide is subjected to saturation mutagenesis. In another embodiment, one or more regions are selected for saturation mutagenesis. In that case, n represents a subset or region of the template polypeptide. For example, when the polypeptide is an antibody, the entire antibody or one or more complementarity determining regions (CDR) of the antibody are subjected to saturation mutagenesis.

The disclosure of the evolution of CPD thus includes mapping methods of a set of mutant antibodies formed from a shortened template antibody having at least one, and preferably six, complementarity determining regions (CDRs), the CDRs comprising set n amino acid residues, the method comprising (a) generating n independent sets of antibodies, each set comprising member antibodies having a predetermined number of different amino acid residues X in a predetermined individual position of the CDR; in which each set of antibodies differs in the predetermined individual position; and the number of different member antibodies generated is equivalent to n x X; (b) test each set to determine at least one predetermined property, characteristic or activity; (c) for each member to identify any change in a property, characteristic or activity in relation to the template polypeptide; and (d) create a structural position map of such changes. For antibodies, the property, characteristic or predetermined property may be binding affinity and / or immunogenicity. As discussed above, as an alternative, an individual population can be generated that comprises all the mutated antibody sets.

In addition, production methods of a set of mutant antibodies formed from a shortened template antibody having at least one complementarity determining region (CDR) are provided, the CDR comprising amino acid residues, the method comprising: (a) generate n independent sets of antibodies, each set comprising member antibodies that have a predetermined number of different amino acid residues at a predetermined individual position of the CDR; in which each set of antibodies differs in the predetermined individual position; and the number of different member antibodies generated is equivalent to n x X. In another embodiment, the antibody comprises six CDRs, and together the CDRs comprise n amino acid residues.

The CPD ™ evolution method includes a functional position map (EvoMap ™) produced by the methods described herein. In a further embodiment, certain residues particularly sensitive to change may be indicated as follows! in the EvoMap ™. Additional optimization can be implemented by making mutation changes by adding positions outside these sensitive positions. It is also possible to use the EvoMap ™ to recognize and recombine beneficial individual amino acid substitutions, and examine to further optimize the desired characteristics in the target molecule, in a procedure called combinatorial protein synthesis (CPS ™).

Combinatorial synthesis of proteins

Combinatorial protein synthesis (CPS ™) involves combining individual matches of CPE, CPI, CPD, or any other evolutionary technique to combine two or more mutations. CPS is used to synthesize antibodies with combined mutations that are then examined to determine optimized gene characteristics and antibodies. A diagram of CPS ™ is shown in Figure 3. In one aspect, two or more 5 point mutations that result in higher mutants or neutral mutations are combined in the cPs.

In one embodiment, CPE is combined with CPS to create mutants, which are examined to detect the desired property. In one aspect, time and resources can be saved in the CPE procedure by changing 2 aa or 3 aa or 4 aa each time against one each time; so that if the number of aa in the antibody is N, the total number generated and examined for 2 aa each time is (2o2) x ^ N; 3 each time it is (203) x1 / 3N, etc. For example, in a specific aspect, (in the example of 2 aa): the 1st aa in the 1st position of aa is combined with the 20 in the 2nd position of aa and the others remain the same, then the 2nd aa in the 1st position of aa with the 20 in the 2nd position of aa and the others remain the same. The entire population is examined for higher mutants and then mutation is performed in the second set of the next two years later. In a similar aspect, this can be done for 3 years at a time or 4 years at a time. In another aspect, the CPE procedure 15 with CPS of higher mutants is optionally followed (including any subset thereof).

In one aspect, unnatural amino acids can be incorporated into the process (so that the other 19 amino acids, or a subset thereof, plus unnatural amino acids) using novel technologies such as the quadrupole codon described in the attached and related articles. Neumann et al. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome Nature 464, 441-444 (February 14, 2010). 20 In this regard, CPE / CPS is performed for the incorporation of unnatural amino acids.

In a further aspect, the entire CPE library is created synthetically (synthesizing all the molecules in commercially available machines). In the case that the machine cannot create large enough strands, fragments are synthesized and then linked to generate full-length molecules. This library is examined and followed with CPS to combine desired mutations. This is a two-stage procedure in which the CPE is followed by CPS, not a single CPE stage.

In another aspect, a CPE library is generated and examined, then followed by combining by higher mutant CPS as follows: if there are 10 higher mutants, an individual molecule with the 10 changes is tested, then all are tested. the versions of 9 mutations, then 8, 7, 6, 5 etc. until one of the groups does not find an improved molecule with respect to any in the previous group. Once an improved molecule is identified, the procedure can be terminated.

In a further aspect, CPE is performed to identify higher mutants and neutral mutations to determine affinity and expression, then CPS is performed with combinations of higher mutants and neutral mutations, and the library is reexamined for additional improvements in features such as function. , affinity and / or expression.

In a further aspect, CPE is performed on codons of the Fc or other domain to detect glycosylation changes.

In another aspect, CPE or CPE can be performed in combination with microRNA CPS or introns.

In a further aspect, CPE or CPE combined with rodent antibody CDR CPS is performed, then examined for higher mutants, followed by humanization.

In one aspect, CPE or CPE combined with CPS is performed to produce alternative intermediate nucleotides that lead to the desired mutation in the final reaction, for example, a methylated cytosine that becomes an uracil.

In one aspect, CPE or CPE combined with more computerized CPS is used to convert mouse CDRs into human CDRs and vice versa.

In one aspect, CPE or CPE combined with CPS with 2 and 3 mutations separated by the entire antibody is used.

In another aspect, CPE or CPE combined with CPS is used in a double chain vector for examination evaluation to detect an increase in sensitivity.

In a further aspect, CPE or CPE combined with CPS is used to select allosteric changes.

Any of several test techniques can be used to evaluate CPE / CPS mutants. In one aspect, CPE or CPE mutants combined with CPS can be secreted and presented in eukaryotic hosts. Alternatively, 50 CPE or CPE mutants combined with CPS in E. coli can be produced and examined in eukaryotic hosts.

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In another aspect, CPE is performed starting from 15 aa or 10 aa and followed by CPS; then followed by the rest of the remaining 19 years. In another aspect, CPE or CPE combined with CPS is used to evolve antibodies specifically with amino acid changes that are not on the surface. In one aspect, CPE can be used for multidimensional epitopic mapping. In another aspect, examination by cPe or CPE combined with CPS may be performed transiently in eukaryotic cells. In a further aspect, CPE or CPE combined with CPS is performed, then sequencing and alignment of all clones is performed, for example, in a chip-based or well-based format for expression and examination. In another aspect, CPE or CPE combined with CPS is used to evolve metal ion coordination by selecting varying ion concentrations. In a further aspect, CPE or CPE combined with CPS is performed, and antibodies are expressed and examined in cell-free conditions and in non-human living organisms. In one aspect, the examination is performed by means of CPE or CPE combined with stem cell CPS to determine variable effects on the differentiation and expression of antibodies and RNA and mRNA. In a further aspect, the multiplex examination is performed by means of CPE or CPE combined with CPS to detect multiple antibody characteristics such as expression and binding. In another aspect, CPE or CPE combined with CPS is performed in template molecules that participate in brain transport and membrane crossing; and mutants are examined for improved characteristics. In one aspect, CPE or CPE mutants combined with CPS are examined to detect hygroscopic antibody characteristics. In another aspect, CPE or CPE mutants combined with CPS are tested for dynamic antibodies. In one aspect, the examination is performed by means of CPE or CPE combined with CPS outside the objective condition to identify mutants within the objective condition and vice versa.

In one embodiment, any of the above aspects of CPE or CPE combined with CPS are used in combination with a selected method of CPT, CPD, and combination of CPD with CPI.

In another embodiment, any of the above aspects of CPE or CPE combined with CPS is used in combination with a selected method of flexible evolution and synergistic evolution made from a mold.

The term "template" may refer to a base polypeptide or a polynucleotide encoding such a polypeptide. As one skilled in the art would appreciate, any mold can be used in the methods and compositions of the present invention. Molds can be used that can be mutated and thereby evolved to guide the synthesis of another polypeptide or polypeptide library as described in the present invention. As described in more detail herein, the template that can be evolved codes for the synthesis of a polypeptide and can subsequently be used to decode the history of the synthesis of the polypeptide, to indirectly amplify the polypeptide, and / or to make it evolve. (i.e., diversify, select and amplify) the polypeptide. The mold that can be evolved is, in certain embodiments, a nucleic acid. In a certain embodiment of the present invention, the template is based on a nucleic acid. In other embodiments, the template is a polypeptide.

The nucleic acid templates used in the present invention are composed of DNA, RNA, a DNA and RNA hybrid, or a derivative of DNA and RNA, and can be single or double stranded. The template sequence is used to code for the synthesis of a polypeptide, preferably a compound that is not, or does not resemble, a nucleic acid or nucleic acid analog (for example, an unnatural polymer or a small molecule). In the case of certain unnatural polymers, the nucleic acid mold is used to align the monomer units in the sequence in which they appear in the polymer and place them in close proximity to adjacent monomer units along the mold so that They will react and bind through a covalent bond. In certain other embodiments, the template can be used to generate unnatural polymers by PCR amplification of a DNA template library consisting of a random region of nucleotides.

It will be appreciated that the mold can vary greatly in the number of bases. For example, in certain embodiments, the mold may be 10 to 10,000 bases in length, preferably between 10 and 1,000 bases in length. The length of the mold will of course depend on the length of the codons, the complexity of the library, the length of the unnatural polymer to be synthesized, the complexity of the small molecule to be synthesized, the use of spacer sequences, etc. The nucleic acid sequence can be prepared using any method known in the art to prepare nucleic acid sequences. These methods include both in vivo and in vitro methods including pCr, plasmid preparation, endonuclease digestion, solid phase synthesis, in vitro transcription, strand separation, etc. In certain embodiments, the nucleic acid template is synthesized using an automated DNA synthesizer.

As discussed above, in certain embodiments of the invention, the method is used to synthesize polypeptides that are not, or do not resemble, nucleic acids or nucleic acid analogs. Therefore, in certain embodiments of the present invention, the nucleic acid template comprises base sequences encoding the synthesis of an unnatural polymer or small molecule. The message encoded in the nucleic acid template preferably begins with a specific codon that takes place in a chemically reactive site from which polymerization can take place, or in the case of synthesizing a small molecule the "initiation" codon can encode for an anti-codon associated with a small molecule shell or a first reactant. The "initiation" codon of the present invention is analogous to the "initiation" codon, ATG, which encodes

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for the amino acid methionine.

In still other embodiments of the invention, the nucleic acid template itself may be modified to include an initiation site for polymer synthesis (eg, a nucleophile) or a small molecule framework. In certain embodiments, the nucleic acid template includes a hairpin loop at one of its ends that ends in a reactive group used to initiate polymerization of the monomer units. For example, a DNA template may comprise a hairpin loop that ends in a 5 'amino group, which may or may not be protected. Polymerization of the unnatural polymer can begin from the amino group. The reactive amino group can also be used to bond a small molecule framework on the nucleic acid template in order to synthesize a small molecule library.

To terminate the synthesis of the unnatural polymer, a "termination" codon must be included in the nucleic acid template preferably at the end of the coding sequence. The "termination" codon of the present invention is analogous to the "termination" codons (ie, TAA, TAG, TGA) found in mRNA transcripts. These codons lead to the termination of antibody synthesis. In certain embodiments, a "termination" codon is chosen that is compatible with the artificial genetic code used to code for the unnatural polymer. For example, the "termination" codon must not conflict with any other codon used to code for the synthesis, and must be of the same general format as the other codons used in the mold. The "termination" codon can code for a monomer unit that terminates the polymerization by not providing a reactive group for further binding. For example, a terminating monomer unit may contain a blocked reactive group such as an acetamide instead of a primary amine. In still other embodiments, the terminator monomer unit comprises a biotinylated terminal end that provides a convenient mode of termination of the polymerization and purification step of the resulting polymer.

In one embodiment, mutagenized DNA products are used directly as a template for in vitro synthesis of the corresponding mutant antibodies. Due to the high efficiency with which substitutions of the 19 amino acids can be generated in an individual residue, it is possible to perform saturation mutagenesis in numerous residues of interest, either independently or in combination with other mutations within the antibody. As used herein, "full saturation" mutagenesis is defined as replacing a given amino acid within an antibody with the other 19 naturally occurring amino acids. For example, genetic site saturation mutagenesis, which systematically explores all possible substitutions of individual amino acids throughout an antibody sequence systematically, is disclosed in Kretz et al., Methods in Enzymology, 2004, 388 : 3-11; Short, U.S. Patent No. 6,171,820; and Short, U.S. Patent No. 6,562,594.

In one aspect, this invention provides for the use of codon primers (containing a degenerate N, N, G / T sequence) to introduce point mutations into a polynucleotide, so that a set of progeny polypeptides is generated in which a The full range of individual amino acid substitutions is represented at each amino acid position (see U.S. Patent No. 6,171,820; see also U.S. Patent No. 5,677,149). The oligos used are contiguously composed of a first homologous sequence, a degenerated N, N, G / T sequence, and preferably but not necessarily a second homologous sequence. The translation products of the posterior progeny from the use of such oligos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneration of the N, N, G / T sequence includes codons for the 20 amino acids.

The use of codons is one of the important factors in eukaryotic gene expression. The frequencies with which different codons are used vary significantly between different hosts, and between antibodies expressed at high or low levels within the same organism. The most likely reason for this variation is that preferred codons correlate with the abundance of related tRNAs available within the cell. It is possible that codon usage and tRNA acceptor concentrations have evolved together, and the selection pressure for this coevolution is more pronounced for highly expressed genes than for genes expressed at low levels.

In one aspect, a degenerate oligo of this type (consisting of a degenerate N, N, G / T cassette) is used to subject each original codon in a parental polynucleotide template to a full range of codon substitutions. In another aspect, at least two degenerate N, N, G / T cassettes are used, either in the same or not oligo, to subject at least two original codons in a parental polynucleotide template to a full range of substitutions of codons Therefore, more than one N, N, G / T sequence may be contained in an oligo to introduce amino acid mutations in more than one site. This plurality of N, N, G / T sequences may be directly contiguous, or be separated by one or more additional nucleotide sequence (s). In another aspect, usable oligos can be used to introduce additions and deletions either alone or in combination with the codons containing an N, N, G / T sequence, to introduce any combination or permutation of additions, deletions and / or substitutions of amino acids.

In another aspect, the present invention provides the use of degenerate cassettes that have less degeneration.

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than the sequence N, N, G / T. For example, it may be desirable in some cases to use (for example, in an oligo) a degenerate triplet sequence consisting of only one N, wherein said N may be in the first, second or third position of the triplet. Any other base including any combination and permutations thereof can be used in the remaining two positions of the triplet. Alternatively, it may be desirable in some cases to use (eg, in an oligo) a degenerate N, N, N triplet sequence.

However, it is appreciated that the use of a degenerate N, N, G / T triplet as disclosed herein is advantageous for several reasons. In one aspect, this invention provides a means to systematically and fairly easily generate the replacement of the full range of possible amino acids (for a total of 20 amino acids) in each and every amino acid position in a polypeptide. Therefore, for a 100 amino acid polypeptide, the present invention provides a way to systematically and fairly easily generate 2000 different species (ie, 20 possible amino acids per position X 100 amino acid positions). It is appreciated that, through the use of an oligo containing a degenerate N, N, G / T triplet, 32 individual sequences encoding 20 possible amino acids are provided. Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using such an oligo, 32 different progeny polynucleotides are generated that encode for 20 different polypeptides. In contrast, the use of a non-degenerated oligo in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.

Thus, in a preferred embodiment, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules so that the 20 amino acids are represented in a specific amino acid position corresponding to the mutagenized codon position. in the parental polynucleotide. Degenerated progeny polypeptides 32 times generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (eg, cloned into a suitable E. coli host using an expression vector) and subjected to expression examination. When an individual progeny polypeptide having a change in a property (as compared to the template polypeptide) is identified by examination, it can be sequenced to identify the amino acid substitution responsible for such a change contained therein.

Any recombinant mutagenic or chemical synthesis method can be used to generate the population of mutant polypeptides. The practice of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are explained in more detail in the bibliography. See, for example, Molecular Cloning A Laboratory Manual, 2nd ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Cloning DNA, volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al., U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treaty, Methods in Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Cabs eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, volumes 154 and 155 (Wu et al. Eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walquer, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embiyo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In one embodiment, the template polypeptide is an antibody. The antibody is subjected to the methods described herein to, for example, map and understand which positions within the CDR effect binding affinity. Techniques for preparing and using various antibody-based constructs and fragments thereof are well known in the art. An important aspect of the present invention is the identification of residues that play, or are likely to play, a role in the interaction of interest (eg, antigen-antibody interaction, metal chelation, receptor binding, substrate binding, etc. .). Any antibody or antibody fragment according to the present invention can be used.

In one embodiment, any of the evolution CPE, CPI, CPD and CPS platforms can be used to generate agonist antibodies, ie activating antibodies. These evolution technologies allow the generation of agonist antibodies beyond the activation of the simplest type of reticulation of proteins and in particular allow the activation of receptors such as GPL-1 or 2 that are traditionally activated by peptides.

In one aspect, antibodies are selected by FACS or microscope or equivalent to detect weakly activating antibodies using cells with fluorescent signals that fluoresce when the cell surface receptor is activated. Subsequently, the evolution tools are used to enhance this activation. The CPS technology is then used to combine superior mutants.

In another aspect, an antibody that binds to the receptor activation site is selected as determined by epltopos mapping. CPE, CPI and / or CPD techniques are used to select mutants that cause

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receptor stimulation as determined by an intracellular reading such as fluorescence in response to the release of calcium ions or other assays that are well known in the art. The CPS technology is then used to combine superior mutants.

In a particular aspect, some of the key advantages of CPI with single, double or triple amino acid inserts are that these inserted amino acids can be extended into the receptor binding cavity to activate the receptor. In another particular aspect, the CPD can reshape and / or reposition amino acids that interact with the receptor to improve or effect activation and finally the CPE can make relatively smaller changes to effect the activation of the receptor.

The specificity of an antibody is determined by the complementarity determining regions (CDR) within the light chain variable regions (VL) and heavy chain variable regions (VH). The Fab fragment of an antibody, which is approximately one third of the size of a complete antibody contains the heavy and light chain variable regions, the entire light chain constant region and a part of the heavy chain constant region. Fab molecules are stable and are well associated due to the contribution of constant region sequences. However, the performance of functional Fab expressed in bacterial systems is lower than that of the smaller Fv fragment that contains only the variable regions of the heavy and light chains. The Fv fragment is the smallest part of an antibody that still retains a functional antigen binding site. The Fv fragment has the same binding properties as the Fab, however, without the stability conferred by the counting regions, the two Fv chains can be relatively easily dissociated under dilute conditions.

In one aspect, VH and VL regions can be fused by means of a polypeptide linker (Huston et al., 1991) to stabilize the antigen binding site. This individual polypeptide Fv fragment is known as a single chain antibody (scFv). The VH and VL can be arranged with any domain in the first place. The linker joins the carboxyl-terminal end of the first chain with the amino-terminal end of the second chain.

One skilled in the art will recognize that Fv single chain, heavy or light chain Fv or Fab fragments can be used with this system. A heavy or light chain can be mutagenized followed by the addition of the complementary chain to the solution. The two chains are then allowed to combine and form a functional antibody fragment. The addition of random non-specific heavy or light chain sequences allows the production of a combinatorial system to generate a library of various members.

Generally, a single chain expression polynucleotide is generated. This expression polynucleotide contains: (1) a single chain antibody cassette consisting of a Vh domain, a spacer peptide and a Vl domain operably linked to encode for a single chain antibody, (2) a promoter suitable for transcription in vitro (eg, T7 promoter, SP6 promoter, and the like) operably linked to ensure in vitro transcription of the single chain antibody cassette that forms an mRNA encoding a single chain antibody, and (3) a sequence of termination of transcription suitable to function in an in vitro transcription reaction. Optionally, the expression polynucleotide may also comprise an origin of replication and / or a selectable marker. An example of a suitable expression polynucleotide is pLM166.

The Vh and Vl sequences can be conveniently obtained from a library of Vh and Vl sequences produced by PCR amplification using primers specific to the V gene family or primers specific to the V gene (Nicholls et al., J. Immunol. Met., 1993, 165: 81; WO93 / 12227) or are designed according to conventional methods known in the art based on the sequence information available. Normally, VH and VL sequences of mouse or human being are isolated. The VH and VL sequences are then ligated, usually with an intermediate spacer sequence (for example, encoding a flexible peptide spacer in frame), forming a cassette encoding a single chain antibody. Normally, a library comprising a plurality of Vh and Vl sequences (sometimes also with a plurality of spacer peptide species represented) is used, in which the library is constructed with one or more of the mutated Vh and Vl sequences. to increase sequence diversity particularly in CDR residues, sometimes in framework region residues. Region V sequences can be conventionally cloned as cDNA or PCR amplification products for cells expressing immunoglobulins. For example, human hybridoma cells, or lymphoma, or other cell line that synthesizes inmunglobulin or secreted or cell surface can be used for the isolation of polyA + RNA. RNA is then used for the synthesis of cDNA primed with oligo dT using the enzyme reverse transcriptase (for general methods see Goodspeed et al., Gene 1989, 76: 1; Dunn et al., J. Biol. Chem., 1989, 264: 13057). Once the product of pCr or cDNA from region V is isolated, it is cloned into a vector to form a single chain antibody cassette.

To achieve the construction of antibodies and antibody fragments, the coding genes are isolated and identified. Genes can be modified to allow donation in an expression vector or in vitro transcription / translation. Although methods such as probe DNA testing for VH and

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VL from hybridoma cDNA (Maniatis et al., 1982) or construct a synthetic gene for VH and VL (Barbas et al., 1992), a convenient way is to use template-directed methods to amplify antibody sequences. A diverse population of antibody genes can be amplified from a template sample by designing primers for the sequences conserved at the 3 'and 5' ends of the variable region known as the framework region or for the constant regions of the antibody (Iverson et al. ., 1989). Within the primers, restriction sites can be placed to facilitate donation in an expression vector. By directing the primers to these conserved regions, the diversity of the antibody population is maintained to allow the construction of diverse libraries. The specific species and class of antibody can be defined by the selection of the primer sequences as illustrated by the large number of sequences for all types of antibodies provided in Kabat et al., 1987.

Messenger RNA isolated from the spleen or peripheral blood of an animal can be used as a template for amplification of an antibody library. Under certain circumstances, when it is desirable to present a homogeneous population of antibody fragments on the cell surface, mRNA can be isolated from a population of monoclonal antibodies. Messenger RNA can be prepared from any source by conventional methods and used directly or for the preparation of a cDNA template. The generation of mRNA for antibody cloning purposes is easily accomplished by following well-known procedures for the preparation and characterization of antibodies (see, for example, Antibodies: A Laboratory Manual, 1988).

The generation of monoclonal antibodies (Acm) generally follows the same procedures as those for preparing polyclonal antibodies. In summary, a polyclonal antibody is prepared by immunizing an animal with an appropriate immunogenic composition and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Normally the animal used for the production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Due to the relatively large blood volume of rabbits, rabbits are usually preferred for the production of polyclonal antibodies.

Immunogenic compositions often vary in immunogenicity. It is often necessary, therefore, to strengthen the host's immune system, as can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are Californian Barnacle Hemocyanin (KLH) and Bovine Serine Albumine (BSA). Other albumines such as ovoalbumine, rat serine albumine or rabbit serine albumine can also be used as carriers. Recognized means for conjugating a polypeptide to a carrier protein are well known and include glutaraldehldo, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimides and bis-diazotized benzidine.

The immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific immune response stimulators, known as adjuvants. Exemplary and preferred adjuvants include Freund's complete adjuvant (a non-specific stimulator of the immune response containing inactivated Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies with the nature of the immunogen as! as the animal used for immunization. A variety of vlas can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by taking blood samples from the immunized animal at various time points after immunization. A second, booster injection can also be administered. The reinforcement and titration procedure is repeated until a suitable title is achieved. When a desired level of immunogenicity is obtained, blood can be drawn from the immunized animal and the serum isolated, the spleen stored and collected for mRNA isolation from the polyclonal response or the animal can be used to generate Acm for mRNA isolation. from a homogeneous population of antibodies.

Acm can be easily prepared through the use of well-known techniques, such as those exemplified in US Patent No. 4,196,265. Normally, this technique involves immunizing a suitable animal with a selected immunogen composition, for example a small molecule hapten conjugated to a carrier, a protein, a purified or partially purified polypeptide or peptide. The immunization composition is administered in an effective manner to stimulate antibody producing cells. Rodents such as mice and rats are frequently used animals; However, the use of rabbit, sheep, frog cells is also possible. The use of rats may provide certain advantages (Goding, pages 60-61, 1986), but mice are preferred, particularly the BALB / c mouse since this is the most commonly used routinely and generally provides a higher percentage of stable fusions. .

After immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the Acm generation protocol. These cells can be obtained from spleens, tonsils or lymph nodes that have undergone biopsy, or from blood samples. Spleen cells and blood cells are preferable, the former because they are a rich source of cells

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producers of antibodies that are in the plasmablast phase in division, and the latter because blood can be easily accessed. Often, an animal panel will have been immunized and the animal's spleen will be removed with the highest antibody titer and spleen lymphocytes will be obtained by homogenizing the spleen with a syringe. Normally, a spleen of an immunized mouse contains approximately 5X107 to 2X108 lymphocytes.

The antibody-producing B lymphocytes of the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suitable for use in hybridoma production fusion procedures are preferably not antibody producing, have high fusion efficiency and enzymatic deficiencies that make them unable to grow in certain selective media that support the growth of only fused cells. desired (hybridomas).

Any one of several myeloma cells can be used, as is known to those skilled in the art (Goding, pages 65-66, 1986; Campbell, 1984). For example, when the immunized animal is a mouse, P3- X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45 can be used -GTG 1.7 and S194 / 5XX0 Bul; for rats, R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in relation to human cell fusions.

A preferred murine myeloma cell is the NS-1 myeloma cell line (also called P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting the GM3573 cell line deposit number. Another mouse myeloma cell line that can be used is the non-producing cell line SP2 / 0 of mouse murine myeloma resistant to 8-azaguanine.

Methods for generating myeloma cell hybrids and lymph node cells or antibody producing spleen usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, although the ratio may vary between 20: 1 and 1: 1, respectively. , in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler & Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding pags. 71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, from 1X10-6 to 1X10-8. However, this is not a problem, since viable, fused hybrids differ from parental, non-fused cells (particularly non-fused myeloma cells that normally continue to divide indefinitely) by culture in a selective medium. The selective medium is generally one that contains an agent that blocks de novo nucleotide synthesis in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells that can carry out nucleotide salvage routes can survive in HAT media. Myeloma cells are defective in key enzymes of the salvage pathway, for example, hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can carry out this route, but they have a limited life in culture and generally die within approximately two weeks. Therefore, the only cells that can survive in selective media are hybrids formed from B cells and myeloma.

This culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing the cells by dilution of individual clones in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) to detect the desired reactivity. Simple and rapid tests include radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, punctual immunological binding assays, and the like.

The selected hybridomas are serially diluted and cloned to give individual antibody producing cell lines from which clones can then propagate indefinitely to provide Acm. Cell lines can be used for the production of Acm in two basic modes. A sample of the hybridoma (often in the peritoneal cavity) can be injected into a histocompatible animal of the type that was used to provide the myeloma and somatic cells for the original fusion. The injected animal develops tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, such as serum or ascitic fluid, can then be extracted to provide Acm at high concentration. Individual cell lines could also be cultured in vitro, where the Acm are naturally secreted into the culture medium from which they can be easily obtained in high concentrations. The Acm produced by any means can be further purified, if desired, using filtration, centrifugation and

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various chromatographic methods such as HPLC or affinity chromatography.

After isolation and characterization of the desired monoclonal antibody, mRNA can be isolated using techniques well known in the art and used as a template for amplification of the target sequence.

Several mold-dependent procedures are available to amplify the target sequences before and after mutagenesis. One of the best known amplification methods is the polymerase chain reaction (called PCR) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al. , 1990. In summary, in PCR, two primer sequences are prepared that are complementary to regions in opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphate is added to a reaction mixture together with a DNA polymerase, for example, Taq polymerase. If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to extend along the target sequence by adding nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, the excess primers will bind to the target and the reaction products and the procedure is repeated. Preferably a PCR amplification procedure with reverse transcriptase can be performed in order to quantify the amount of amplified target. Polymerase chain reaction methodologies are well known in the art. Using enzymatic amplification techniques such as PCR, desired control elements can be designed in the primer and therefore incorporated into the DNA product.

Another method for amplification is the ligase chain reaction ("CSF"), disclosed in EPA No. 320 308. In the CSF, two pairs of complementary probes are prepared, and in the presence of the sequence target, each pair will join opposite complementary strands of the target so that they are contiguous. In the presence of a ligase, the two pairs of probes will join to form an individual unit. Through temperature cycles, as in the PCR, the bound linked units are dissociated from the target and then serve as "target sequences" for ligation of excess probe pairs. US Patent No. 4,883,750 describes a method similar to CSF for attaching pairs of probes to a target sequence.

Qbeta replicase, described in PCT application No. PCT / US87 / 00880, can also be used as the amplification method. In this method, an RNA replication sequence is added that has a region complementary to that of a target to a sample in the presence of an RNA polymerase. The polymerase will copy the replication sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve amplification of target molecules containing 5'- [alpha-thio] -nucleotide triphosphates in a strand of a restriction site can also be useful in nucleic acid amplification (Walquer et al., 1992).

Strand shift amplification (SDA) is another method of carrying out isothermal nucleic acid amplification that involves multiple strands of strand displacement and synthesis, that is, translation of nicks. A similar method, called a repair chain reaction (RCR), involves pairing several probes throughout a region selected as a target for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives to achieve easy detection. A similar approach is used in sDa. Specific target sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe that has 3 'and 5' sequences of non-specific DNA and medium sequence of specific RNA is hybridized with DNA that is present in a sample. After hybridization, the reaction with RNaseH is treated, and the products of the probe are identified as distinctive products that are released after digestion. The original mold is paired with another cycling probe and the reaction is repeated.

Other amplification methods are described in GB application No. 2 202 328, and in PCT application No. PCT / US89 / 01025, they can be used according to the present invention. In the first application, "modified" primers are used in a template and enzyme dependent synthesis, similar to pCr. The primers can be modified by labeling with a capture moiety (for example, biotin) and / or a detector moiety (for example, enzyme). In the last request, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is cataloged cleavage. After excision, the intact target sequence to which the excess probe is to be bound is released. Excision of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989). In NASBA, nucleic acids can be prepared for amplification by extraction with conventional phenol / chloroform, thermal denaturation of a clinical sample, lysis buffer treatment and minicentrifugation columns for DNA and RNA isolation or RNA extraction with guanidinium chloride . These amplification techniques involve pairing a primer that has specific target sequences. After polymerization, DNA / RNA hybrids are digested with RNase H while double stranded DNA molecules are thermally denatured again. In any case, single stranded DNA is completely made

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double stranded by the addition of a second specific target primer, followed by polymerization. The double stranded DNA molecules are then transcribed multiplely by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, RNAs are transcribed in reverse to give double stranded DNA, and transcribed again with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate specific target sequences.

Davey et al., EPA No. 329 822 discloses a nucleic acid amplification procedure that involves cyclically synthesizing single stranded RNA ("mRNA"), mDNA, and double stranded DNA (cDNA), which can be used according to present invention. The mRNA is a first template for a first oligonucleotide primer, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the DNA duplex: resulting RNA by the action of ribonuclease H (RNase H, a specific RNA of duplex RNA with either DNA or RNA). The resulting mDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) in 5 'with respect to its homologla to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double stranded DNA molecule ("cDNA"), which has an identical sequence to that of RNA original among the primers and which additionally has, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many copies of DNA RNA. These copies can then be reintroduced into the cycle leading to very rapid amplification. With the appropriate choice of enzymes, this amplification can be done isothermally without the addition of enzymes in each cycle. Due to the cyclic nature of this procedure, it can be chosen that the starting sequence is in the form of either DNA or RNA.

Miller et al., PCT application WO 89/06700 discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter / primer sequence to a single stranded target DNA ("mDNA") followed by transcription of many copies RNA sequence. This scheme is not cyclic, that is, no new molds are produced from the resulting RNA transcripts. Other amplification methods include "race" and "unilateral PCR" (Frohman, 1990; O'Hara et al., 1989).

Methods based on the ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide" can also be used, thereby amplifying the dioligonucleotide, at the amplification stage (Wu et al., 1989).

Amplification products can be analyzed by agarose, agarose acrylamide or polyacrylamide gel electrophoresis using conventional methods (see, for example, Maniatis et al., 1982). For example, a 1% agarose gel taken with ethidium bromide can be used and visualized under UV light. Alternatively, the amplification products can be labeled integrally with radiolabeled or fluorometrically labeled nucleotides. Then gels can be exposed to x-ray film or visualized under the appropriate stimulation spectra, respectively.

The mutagenic methods of the present invention may comprise any mutagenic approach that can be adapted to a particular site in a gene, ie site-specific or site-directed mutagenesis. Because the present invention is based on complete mutagenesis, the present invention contemplates as preferred embodiments mutagenic processes that are fast, efficient and economical.

In one embodiment, the mutagenic method uses chemical synthesis techniques. By doing this, it is possible to place exactly the substitution in one or more particular locations within the gene, and also specifically define the nature of the alterations. Chemical synthesis methods for DNA are well known within the art. Solid phase techniques are preferred in this regard.

An advantage of the solid phase method of genetic synthesis is the opportunity for mutagenesis using combinatorial synthesis techniques. Combinatorial synthesis techniques are defined as the techniques that produce large collections or libraries of compounds simultaneously, sequentially joining different structural units. Libraries can be constructed using free compounds in solution, but preferably the compound binds to a solid support such as a bead, a solid particle or even is presented on the surface of a microorganism.

There are several methods for combinatorial synthesis (Holmes et al., 1995; Burbaum et al., 1995; Martin et al., 1995; Freier et al., 1995; Pei et al., 1991; Bruce et al., 1995; Ohlmeyer et al., 1993), including split or parallel synthesis. Split synthesis can be used to produce small quantities of a relatively large number of compounds, while parallel synthesis will produce larger quantities of a relatively small number of compounds. In general terms, using split synthesis, compounds are synthesized on the surface of a microparticle. At each stage, the particles are divided into several groups for the addition of the next component. The different groups then recombine and divide to form new groups. The procedure is repeated until the compound is complete. Each particle contains several copies of the same compound allowing easy separation and purification. Split synthesis can only be done using a

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solid support.

An alternative technique known as parallel synthesis can be performed either on a solid basis or in solution. Using parallel synthesis, different compounds are synthesized in independent receptacles, often using automation. Parallel synthesis can be performed on a microtiter plate where different reagents can be added to each well in a predefined way to produce a combinatorial library. Parallel synthesis is the preferred approach for use with enzymatic techniques. It will be well understood that there are many modifications of this technique and can be adapted for use with the present invention. Using combinatorial methods, a large number of mutant gene templates can be synthesized.

Mutant genes can also be generated by semisynthetic methods known in the art (Barbas et al., 1992). Using the conserved regions of an antibody fragment as a framework, variable regions can be inserted into random combinations one or more at a time to alter the specificity of the antibody fragment and generate novel binding sites, especially in the generation of antibodies against non-conducive antigens. for immunization such as toxic compounds or labiles. Similarly, a known antibody sequence can be varied by randomly introducing mutations. This can be achieved by methods well known in the art such as the use of error sensitive PCR.

Using the appropriate oligonucleotide primers, PCR is used for the rapid synthesis of the DNA template containing one or more mutations in the binding protein gene. Site-specific mutagenesis is a useful technique in the preparation of individual peptides, or biologically functional equivalent peptides or peptides, through specific mutagenesis of the underlying DNA. The technique also provides an easy ability to prepare and test sequence variants, incorporating one or more of the above considerations, introducing one or more nucleotide sequence changes in the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences encoding the DNA sequence of the desired mutation, as! as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Normally, a primer of 17 to 25 nucleotides in length is preferred, with 5 to 10 residues on both sides of the junction of the sequence being altered.

The technique normally employs a bacteriophage vector that exists in both single and double stranded forms. Useful vectors in site-directed mutagenesis include vectors such as phage M13. These phage vectors are commercially available and are generally well known to those skilled in the art. Double-stranded site directed mutagenesis plasmids are also routinely employed, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single stranded vector, or by fusing two strands of a double stranded vector that includes within its sequence a DNA sequence that encodes the desired protein. An oligonucleotide primer that carries the desired mutated sequence is prepared synthetically. This primer is then paired with the single-stranded DNA preparation, taking into account the degree of mating mismatch when hybridization conditions are selected, and subjected to DNA polymerization enzymes such as Klenow fragment of E. coli polymerase I, with the in order to complete the synthesis of the strand that carries the mutation. Therefore, a heteroduplex is formed in which one strand encodes the original non-mutated sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones that include recombinant vectors that carry the mutated sequence arrangement are selected.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not intended to be limiting, since there are other ways in which sequence variants can be obtained. For example, recombinant vectors encoding the desired gene can be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

In certain applications, substitution of amino acids by site-directed mutagenesis, it is appreciated that less stringent conditions are required. Under these conditions, hybridization can occur even when the probe and target strand sequences are not perfectly complementary, but have mismatches in one or more positions. The conditions can be made less stringent by increasing the salt concentration and lowering the temperature. For example, a condition of medium stringency could be provided by means of 0.1 to 0.25 M NaCl at temperatures of 37 ° C to 55 ° C, while a condition of low stringency could be provided by salt of 0.15 M to 0 , 9 M, at temperatures ranging between 20 ° C and 55 ° C. Therefore, hybridization conditions can be easily manipulated, and therefore will generally be a method of choice depending on the desired results.

In other embodiments, hybridization can be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), KCl

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75 mM, 3 mM MgCl2, 10 mM dithiothreitol, at temperatures between about 20 ° C and 37 ° C. Other hybridization conditions used could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 pM MgCl 2, at temperatures ranging from approximately 40 ° C to 72 ° C. Formamide and SDS can also be used to alter hybridization conditions.

In a particular embodiment, overlap PCR can be employed. In summary, a plasmid is used as a template for the first batch of pCr. The PCR products of the first batch are purified and used, together with external primers, in the overlap extension PCR reaction. The final products contain the site-directed substitution of an amino acid given by all other possible amino acid residues.

The mutant polypeptides generated by the present invention can be characterized using a variety of techniques. In general, protein products can be analyzed to determine the correct apparent molecular weight using SDS-PAGE. This provides an initial indication that the polypeptide, in fact, was synthesized. When compared to the natural molecule, it also indicates whether normal folding or processing is taking place with the mutant. In this regard, it can be verified that it is useful to label the polypeptide. Alternatively, the polypeptide can be identified by gel staining.

Beyond mere synthesis, antibodies can be characterized according to various properties and a wide range of functions. Properties include isoelectric point, thermal stability, sedimentation rate and folding. One way to examine folding is the ability to recognize yourself by a related union partner. The main example of this function is the antibody-antigen interaction. A wide variety of different immunoassay formats are available for this purpose and are well known in the art. Mainly, changes in either affinity or specificity can be determined when the antibody is contacted with a specific antigen or related antigen panels.

Immunoassays can generally be divided into two types: heterogeneous assays that require multiple stages of separation, and homogeneous assays that are performed directly. Heterogeneous immunoassays generally involve an antibody immobilized on a solid matrix. A sample containing a ligand is contacted with the immobilized antibody and the amount of complex formed on the matrix support is determined from a marker directly or indirectly attached to the immobilized complex. As used in the context of the present invention, ligand is defined as a species that interacts with an unidentified molecule forming a stable, tightly bound complex. For practical purposes, the binding affinity is usually greater than 106 M-1 and is preferably in the range of 109-1015 M-1. The ligand can be any of several types of organic molecules, including alicyclic carbohydrates, polynuclear aromatic compounds, halogenated compounds, benzenoids, polynuclear carbohydrates, heterocyclic nitrogen compounds, heterocyclic sulfur compounds, heterocyclic oxygen compounds, and carbohydrates. carbon of alkane, alkene, alkyne, etc. Of particular interest are biological molecules, including amino acids, peptides, proteins, liquids, saccharides, nucleic acids and combinations thereof. Of course it will be understood that these are by way of example only and that immunoassay methods contemplated to detect an extraordinarily wide range of compounds can be applied, provided that an antibody that binds to the ligand of interest can be obtained.

Heterogeneous immunoassays can be performed as sandwich assays in which a molecule of interest is reacted with an immobilized antibody that specifically binds to that molecule with a high affinity. In a second stage, a conjugate formed from the same or different antibody against the antigen and a marker molecule is reacted with the antigen-antibody complex on the immobilization matrix. After removal of the excess free marker conjugate, the bound marker conjugate is measured, which is proportional to the amount of ligand in the sample.

The detection of the formation of immunocomplexes is well known in the art and can be achieved through the application of numerous approaches. These approaches are usually based on the detection of a label or a label, such as any of the radioactive, fluorescent, chemiluminescent, electrochemiluminescent, biological or enzymatic labels or markers known in the art. US patents regarding the use of such markers include US patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Of course, additional advantages can be found through the use of a secondary binding ligand such as a second antibody or a biotin / avidin ligand binding arrangement, as is known in the art.

Preferred methods for detection include radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELlSA), with ELISA being the most preferred due to a general increase in sensitivity. ELISAs are used extensively in biotechnology applications, particularly as immunoassays for a wide range of antigenic substances. The sensitivity of ELISA is based on the enzymatic amplification of the signal.

As used herein, the term "contact" is defined as bringing the components of the reaction to close proximity sufficient to each other to allow the desired interaction to occur. The contacting can be achieved by mixing the components in solution, for example, or by interaction

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heterogeneous such as by flow contact through an immobilization column or matrix that joins one of the components.

Antibodies produced and isolated by the method of the invention are selected to bind to a predetermined target. Normally, the predetermined target will be selected in view of its applicability as a diagnostic and / or therapeutic target. The predetermined target can be a known or unknown epltopo. The antibodies generally bind to a predetermined antigen (eg, the immunogen) with an affinity of at least 1X107 M, preferably with an affinity of at least 5X107 M-1 more preferably with an affinity of at

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minus 1X10 M 'to 1X10 M' or more, sometimes up to 1X10 M 'or more. Frequently, the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), another human biological macromolecule (for example , thrombomodulin, protelna C, carbohydrate antigen, sialized Lewis antigen, L-selectin), or macromolecule associated with non-human disease (eg, bacterial LPS, virion capside prothena or envelope glycoproteln) and the like .

In another example, several reports of the diagnostic and therapeutic utility of scFv have been published (Gruber et al., 1994 op. Cit .; Lilley et al., 1994 op. Cit .; Huston et al., Int. Rev. Immunol 1993, 10: a 195, Sandhu JS, Crit. Rev. Biotechnol., 1992, 12: 437).

High affinity antibodies of the desired specificity can be engineered and expressed in a variety of systems. For example, scFv have been produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861) and can easily be prepared in prokaryotic systems (Owens RJ and Young RJ, J. Immunol. Met., 1994,168 : 149; Johnson S and Bird RE, Methods Enzymol., 1991, 203: 88). In addition, single chain antibodies can be used as the basis for constructing complete antibodies or various fragments thereof (Kettleborough et al., Euro J. Immunol., 1994, 24: 952). The sequence coding for the variable region can be isolated (for example, by PCR amplification or subcloning) and subjected to splicing to give a sequence coding for a desired human constant region to code for a human sequence antibody more suitable for therapeutic uses in humans where immunogenicity is preferably minimized. The polynucleotide (s) having the completely human coding sequence (s) can be expressed in a host cell (for example, from an expression vector in a eukaryotic cell ) and purified to give a pharmaceutical formulation.

The DNA expression constructs will normally include an expression control DNA sequence operatively linked to the coding sequences, including naturally associated heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems into vectors that can transform or transfect eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained in suitable conditions for high-level expression of the nucleotide sequences, and the collection and purification of the mutated "engineered genetic" antibodies.

As stated above, the DNA sequences will be expressed in hosts after the sequences have been operatively linked to an expression control sequence (i.e. placed to ensure transcription and translation of the structural gene). These expression vectors can usually be replicated in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Commonly, expression vectors will contain selection markers, for example, tetracycline or neomycin, to allow detection of transformed cells with the desired DNA sequences (see, for example, U.S. Patent No. 4,704,362).

In addition to eukaryotic microorganisms such as yeasts, cell cultures of mammalian tissues can also be used to produce the polypeptides of the present invention (see, Winnacker, "From Genes to Clones", VCH Publishers, N.Y., N.Y. (1987)). Eukaryotic cells are preferred, because several suitable host cell lines that can secrete intact immunoglobulins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, B cells or hybridomas. Expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 1986, 89: 49), and processing sites of necessary information, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. The sequence coding for the variable region can be isolated (for example, by PCR amplification or subcloning) and subjected to splicing to give a sequence coding for a desired human constant region to code for a human sequence antibody more suitable for therapeutic uses in humans where immunogenicity is preferably minimized. The polynucleotide (s) having the completely human coding sequence (s) can be expressed in a host cell (for example, from an expression vector in a eukaryotic cell ) and purified to give a pharmaceutical formulation.

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The DNA expression constructs will normally include an expression control DNA sequence operatively linked to the coding sequences, including heterologous or naturally associated promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems into vectors that can transform or transfect eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained in suitable conditions for high-level expression of the nucleotide sequences, and the collection and purification of the mutated "engineered genetic" antibodies.

As stated above, the DNA sequences will be expressed in hosts after the sequences have been operatively linked to an expression control sequence (ie, located to ensure transcription and translation of the structural gene). These expression vectors can usually be replicated in host organisms either as episomes or as an integral part of the host's chromosomal DNA. Commonly, expression vectors will contain selection markers, for example, tetracycline or neomycin, to allow detection of transformed cells with the desired DNA sequences (see, for example, U.S. Patent No. 4,704,362, which is incorporated herein by reference).

In addition to eukaryotic microorganisms such as yeasts, cell cultures of mammalian tissues can also be used to produce the polypeptides of the present invention (see, Winnacker, "From Genes to Clones", VCH Publishers, N.Y., N.Y. (1987)). Eukaryotic cells are preferred, because several suitable host cell lines that can secrete intact immunoglobulins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, B cells or hybridomas. Expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 1986, 89: 49), and processing sites of necessary information, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like.

The transcription of eukaryotic DNA can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting sequences of between 10 and 30 bp that increase transcription by a promoter. Enhancers can effectively increase transcription when they are either 5 'or 3' with respect to the transcription unit. They are also effective if they are located within an intron or within the coding sequence itself. Normally, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers and adenovirus enhancers. Also used are mammalian system enhancer sequences, such as the mouse immunoglobulin heavy chain enhancer.

Mammalian expression vector systems will also normally include a selectable marker gene. Examples of suitable markers include the dihydrofolate reductase (DHFR) gene, the thymidine kinase (TK) gene or prokaryotic genes that confer drug resistance. The first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium. Transformed cells can then be identified by their ability to grow on non-complementary media Examples of useful prokaryotic drug resistance genes as markers include genes that confer resistance to G418, mycophenolic acid and hygromycin.

Vectors containing the DNA segments of interest can be transferred to the host cell by well known methods, depending on the type of host cell. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, lipofection or electroporation can be used for other host cells. Other methods used to transform mammalian cells include the use of polybrene, fusion of protoplasts, liposomes, electroporation and microinjection (see, in general, Sambrook et al., Cited above).

Once expressed, antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to conventional methods of the art, including precipitation with ammonium sulfate, fractionation column chromatography, electrophoresis in gel and the like (see, in general, Scopes, R., Protein Purification, Springer-Verlag, NY (1982)). Once purified, partially or even homogeneity if desired, the polypeptides can then be used therapeutically or in the development and performance of test procedures, immunofluorescent stains, and the like (see, in general, Immunological Methods, volumes I and II, Eds. Lefkovits and Pernis, Academic Press, NYNY (1979 and 1981)).

The oligopeptides of the present invention can be used for diagnosis and therapy. By way of illustration and not limitation, antibodies can be used to treat cancer, autoimmune diseases or viral infections. For the treatment of cancer, the antibodies will normally bind to an antigen preferentially expressed in cancer cells, such as erbB-2, CEA, CD33, and many other antigens well known to those skilled in the art.

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technique. For the treatment of autoimmune disease, the antibodies will normally bind to an antigen expressed in T cells, such as CD4, the IL-2 receptor, the various T cell antigen receptors and many other antigens well known to those skilled in the art. technique (for example, see Fundamental Immunology, 2nd ed., WE Paul, ed., Raven Press: New York, NY). For the treatment of viral infections, the antibodies will normally bind to an antigen expressed on cells infected by a particular virus such as the various glycoprotelns (eg, gB, gD, gE) of herpes simplex virus and cytomegalovirus, and many other antigens well known to those skilled in the art (for example, see Virology, 2nd ed., BN Campos et al., eds., (1990), Raven Press: New York, NY).

Pharmaceutical compositions comprising antibodies of the present invention are useful for parenteral administration, that is, subcutaneously, intramuscularly or intravenously. Compositions for parenteral administration will commonly comprise a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the mutant antibodies in these formulations can vary widely, that is, from less than 0.01%, usually at least 0.1% to as much as 5% by weight and will be selected primarily based on volumes, viscosities of fluids, etc., according to the particular administration mode selected.

Therefore, a typical pharmaceutical composition for intramuscular injection could be constituted to contain 1 ml of sterile buffered water, and 1 mg of mutant antibody. A typical intravenous infusion composition may be constituted to contain 250 ml of sterile Ringer's solution, and 10 mg of mutant antibody. Those skilled in the art will know or find obvious real methods for preparing compositions that can be administered parenterally and are described in more detail in, for example, Remington's Pharmaceutical Science, 20th Ed., Mack Publishing Company, Easton, Pa. (2000 ).

Manufacture of candidates and / or evolved candidates

In one embodiment, the eukaryotic system is a mammalian system selected from one of the group consisting of CHO, HEK293, IM9, DS-1, THP-1, Hep G2, COS, NIH 3T3, C33a, A549, A375 cell lines. SK-MEL-28, DU 145, PC-3, HCT 116, Mia PACA-2, ACHN, Jurkat, MM1, Ovcar 3, HT 1080, Panc-1, U266, 769P, BT-474, Caco-2, HCC 1954, MDAMB-468, LnCAP, NRK-49F and SP2 / 0; and mouse splenocytes and rabbit PBMC.

In one embodiment, a wide variety of mammalian host cells can be used in the manufacture of the candidate, including fibroblast cells (3T3, mouse; BHK21, Syrian hamster), epithelial cells (MDCK, dog; Hela, human; PtK1, rat kangaroo), plasma cells ((SP2 / 0 and NS0, mouse), kidney cells (293, human; COS, monkey), ovarian cells (CHO, Chinese hamster), embryonic cells (R1 and E14,1, mouse ; H1 and H9, human being; PER C.6, human being).

In certain aspects, recombinant antibodies are produced in CHO and NS0 and SP2 / 0 cell lines. In a specific aspect, the mammalian system is a CHO-S cell line. The most frequently used expression vector systems are glutamine synthetase expression systems and others based on dihydrofolate reductase genes.

In another embodiment, the eukaryotic system is a system of yeast cells. In one aspect, the yeast cell system is selected from S. cerevisiae or Picchia cells.

In the method of the present invention, the hosts used to examine evolved molecules are the same as the hosts used for making matches. In another aspect of the present invention, the genetic system used for the discovery and evolution of antibodies is exactly the same as the genetic system used to make the antibody for commercial applications.

Biosimilar drugs

Biosimilar drugs are therapeutic products based on protein that have an identical amino acid sequence (i.e. chemical composition) than an approved ethical drug that is no longer protected by a patent. In one aspect, the CIAO method is particularly relevant for biosimilar drugs. Although it is essential to produce the therapeutic antibody in an equivalent formulation and composition, to be competitive in the market, the biosimilar drug must be manufactured as quickly and economically as possible. The means of

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Cell culture and the development of procedures are some of the most expensive and time-consuming parts of the preparation and production of a biosimilar drug.

The change of the silent mutation codons within a therapeutic antibody changes the codon used for the translation of the protein but retains the amino acid sequence within the antibody. These codon changes in a variety of positions within a molecule, particularly at the amino-terminal end can have a significant impact on expression and in some cases even glycosylation. In one aspect, the CIAO method is used to select and evolve silent mutation codons in an antibody within a host cell similar to the one ultimately used for fabrication. Therefore, the processing time can be reduced due to the higher antibody yields and the fact that the antibody was selected to be expressed in the host cell line, so that most of the traditional manufacturing problems have been ruled out of the molecule . In addition, by selecting the molecules in cheap, serum-free culture media, molecules with codons that allow cheap manufacturing and purification can be selected.

Without further elaboration, it is believed that one skilled in the art can use, using the description above, the present invention to its fullest extent. The following examples should be considered illustrative and therefore are not limiting the rest of the disclosure in any way at all.

Examples

Example 1. Generation and examination of an antibody library

This example describes the method of generating and examining a human antibody library with presentation on the surface of mammalian cells to isolate human antibodies with binding activity to a target antigen using the combination of flow cytometric classification and ELISA.

Library exam by flow cytometric analysis

1. Generate libraries of human antibodies stably integrated into mammalian cells as described in Appendices 1.2, 1.3 and 1.4 below.

2. Expand stable fully human antibody library clones before flow cytometric analysis.

3. On the day of the flow cytometric analysis, wash 1 x 107 cells with 1 x PBS.

4. Detach the cells with Detachin cell detachment medium and collect the cells in 1 x PBS.

5. Centrifuge the cells at 3000 rpm for 5 minutes. Remove the supernatant.

6. Resuspend the cell pellet in 1 ml of 1 x fried PBS and centrifuge at 3000 rpm for 5 minutes.

7. Remove the supernatant and resuspend the cell pellet in 500 ml of 2 mg / ml of purified human protelna 001 in 1 x fried PBS.

8. Incubate on ice for 1 hour with occasional mixing by hand.

9. Centrifuge the cells at 3000 rpm for 5 minutes. Remove the supernatant.

10. Resuspend the cell pellet in 1 ml of 1 x fried PBS and centrifuge at 3000 rpm for 5 minutes.

11. Repeat steps 7 and 8.

12. Remove the supernatant and resuspend the cell pellet in 500 ml of 1 ng / ml of rabbit anti-001 polyclonal antibody in 1 x PBS fried with 10% goat serum.

13. Incubate on ice for 30 minutes with occasional mixing by hand.

14. Centrifuge the cells at 3000 rpm for 5 minutes. Remove the supernatant.

15. Resuspend the cell pellet in 1 ml of 1 x fried PBS and centrifuge at 3000 rpm for 5 minutes.

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16. Repeat steps 7 and 8.

17. Remove the supernatant and resuspend the cell pellet in 500 ml of FITC-conjugated goat anti-rabbit antibody and human goat anti-Fc antibody conjugated with phycoerythrin in 1 x cold PBS with 10% goat serum.

18. Incubate on ice for 30 minutes with occasional mixing by hand.

19. Centrifuge the cells at 3000 rpm for 5 minutes. Remove the supernatant.

20. Resuspend the cell pellet in 1 ml of 1 x cold PBS and centrifuge at 3000 rpm for 5 minutes.

21. Repeat steps 7 and 8.

22. Remove the supernatant and resuspend the cell pellet in 1 ml of 1 x cold PBS with 2% goat serum.

23. Continue with the flow cytometric analysis using Dako MoFlo.

24. Prepare a classification window to include the top 0.1% of the total cells in terms of the PE / FITC fluorescence ratio. Collect the cells that are inside the classification window in 96-well plates with 100 ml of growth media.

Recovery of light chain and heavy chain variable region sequences

1. Expand the clones from 96-well plates to 6-well plates. When the cells reach 80% confluence in the 6-well plates, continue to isolate genomic DNA using the Qiagen DNeasy Tissue kit.

2. Remove the media from the cells by aspiration. Add 500 ml of 1 x PBS to each 6 wells. Scrape plate cells with sterile pipette tips. Transfer the scraped cells in PBS to a sterile microcentrifuge tube.

3. Centrifuge the cells for 5 minutes at 3000 rpm.

4. Remove the supernatant and resuspend the cell pellet in 200 ml of 1 x PBS.

5. Add 20 ml of proteinase K and 200 ml of AL buffer to the sample, mix thoroughly by stirring with vortex and incubate at 56 ° C for 10 minutes.

6. Add 200 ml of ethanol to the sample and mix thoroughly by stirring with vortex.

7. Pipette the mixture from step 6 into a spin column. Centrifuge at 8000 rpm for one minute. Discard the flow through.

8. Add 500 ml of AW1 buffer and centrifuge for one minute at 8000 rpm. Discard the flow through.

9. Add 500 ml of AW2 buffer and centrifuge for 2 minutes at 14,000 rpm. Discard the flow through. Centrifuge again for one minute at 14,000 rpm. Make sure the membrane is completely dry.

10. Place the centrifuge column in a sterile microcentrifuge tube and pipette 200 ml of AE buffer directly onto the membrane.

11. Incubate at room temperature for one minute and centrifuge for one minute at 8000 rpm to elute the genomic DNA.

12. Submit the genomic DNA to QC by preparing the following reactions in 1.5 ml microcentrifuge tubes:

5 ml cDNA

10x sample loading buffer 5 ml___________________________

Total volume 10 ml

Load on a 0.8% agarose gel - TAE ethidium bromide 0.5 mg / ml. Use molecular size marker of

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1 kb DNA as standard. Run the gel at 100 V for 20-30 minutes in 1X TAE buffer.

13. Prepare the following PCR reactions in sterile PCR tubes:

1 ml gDNA

2x HotStar Taq 12.5 ml master mix

Direct variable domain primer * 0.5 ml Inverse variable domain primer * 0.5 ml

H2O ____________________________ 10.5 ml

Total volume_____________________25 ml

* see appendix 1.2 ____________________

14. Place the PCR tubes in the thermal cycler and start the cycling program. Initial activation stage: 15 minutes, 95 ° C, 3-stage cycling

Denaturation: 40 seconds, 94 ° C

Mating: 40 seconds, 55 ° C

Extension: 2 minutes, 72 ° C

Number of cycles: 30

Final extension stage: 10 minutes, 72 ° C

15. Submit the PCR reactions to QC by preparing the following reactions in 1.5 ml microcentrifuge tubes:

PCR reaction 5 ml

10x sample loading buffer 5 ml___________________________

Total volume 10 ml

Load on a 1% agarose gel - TAE ethidium bromide 0.5 mg / ml. Use 1 kb DNA molecular size marker as a standard. Run the gel at 100 V for 20-30 minutes in 1X TAE buffer.

16. Prepare the following cloning reactions in 1.5 ml microcentrifuge tubes using Invitrogen TOPO 2.1 kit:

PCR reaction 4 ml

1 ml saline solution

TOPO vector __________________1ml

Total volume 6 ml

17. Mix the reactions gently and incubate for 5 minutes at room temperature.

18. Add 2 ml of the TOPO cloning reaction from step 17 into a vial of chemically competent E. Shot coli and mix gently.

19. Incubate on ice for 30 minutes.

20. Subject the cells to thermal shock for 30 seconds at 42 ° C.

21. Transfer the tubes to ice and incubate for 2 minutes.

22. Add 250 ml of S.O.C. at room temperature.

23. Stir the tubes horizontally at 37 ° C for one hour at 200 rpm.

24. Spread 10 ml of the transformation on a newly heated LB-carbenicillin plate.

25. Incubate the plate overnight at 37 ° C.

26. Collect 6 clones of each transformation for sequencing.

27. Analyze the heavy chain and light chain variable region sequences. Continue to the second round of exam using the ELISA method.

Digest vector and clones of human antibodies with restriction enzymes

5 The reactions will depend on the restriction enzyme (s) chosen and according to the manufacturer's instructions; Examples are given herein: Prepare the following digestion reactions in a microcentrifuge tube on ice:

Vector DNA (2 mg) x ml

10x Rest Enz buffer x 10 ml

Nuclease-free water c.s. up to 97 ml

Rest Enz 1 (10 U / ml) 3 ml

Rest Enz 2 (10 U / ml) __________________ 3_ml_________

Total reaction volume 100 ml

Human antibody clones (5 ug) x ml 10X Rest Enz buffer x 10 ml

Nuclease-free water c.s. up to 97 ml

Rest Enz 1 (10 U / ml) 3 ml

Rest Enz 2 (10 U / ml) __________________ 3_ml_________

Total reaction volume 100 ml

1. Mix gently and centrifuge briefly (5 s) in microcentrifuge.

2. Incubate the reaction at 37 ° C overnight

10 Digest using CIP vector and purify with the QIAquick PCR purification kit

3. Add 2 ml of Apex phosphatase to the microcentrifuge tube containing the digestion reaction of the vector.

4. Incubate at 37 ° C for 10 minutes.

5. Heat at 70 ° C for 5 minutes to inactivate the Apex phosphatase.

6. Add 500 ml of PBI buffer to the microcentrifuge.

15 7. Mix by vortex and centrifuge quickly.

8. Load 750 ml each time in a column.

9. Centrifuge at 12,000 x g for 1 minute and decant the liquid from the collection tube.

10. Repeat until the entire sample has been processed.

11. Wash with 750 ml of PE buffer (with ethanol added).

20 12. Centrifuge at 12,000 x g for 1 minute and decant the liquid from the collection tube.

13. Place the column again on the collection tube and centrifuge again.

14. Place the column on new microcentrifuge tubes and elute with 50 ml of EB buffer.

Gel purify clones of human antibodies digested with restriction enzymes

1. Prepare the following reactions in a 1.5 ml microcentrifuge tube:

Fully human antibody clones digested with Rest Enz 100 ml

10x sample loading buffer___________________________________3_jml________

Total volume 103 ml

2. Load on a 1% agarose gel -TAE 0.5 mg / ml ethidium bromide. Use 1 kb DNA molecular size marker as a standard. Run the gel at 100 V for 20-30 minutes in 1X TAE buffer.

4. Add 3 volumes of QG buffer to 1 gel volume.

5. Incubate at 50 ° C for 10 minutes until the gel piece has completely dissolved. Add 1 volume 5 of isopropanol gel to the sample and mix.

6. Place a QIAquick centrifuge column in a 2 ml collection tube provided.

7. Apply the sample to the QIAquick column and centrifuge for 1 minute.

8. Discard the flow through and replace the QIAquick column in the same collection tube.

9. Add 0.75 ml of PE buffer to the QIAquick column and centrifuge for 1 minute.

10 10. Discard the flow through and centrifuge the QIAquick column for an additional 1 minute at 17,900 x g

(13,000 rpm).

11. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube.

12. Add 52 ml of EB buffer to the center of the QIAquick membrane and centrifuge the column for 1 minute. Let the column stand for 1 minute and then centrifuge for 1 minute.

15 Bind the variable domain of human HC and LC in digested vector DNA

Prepare the following ligation reaction in a microcentrifuge tube on ice:

Digested vector DNA (100 ng) x ml

Variable domain of human HC and LC and ml

5X 4 ml T4 ligase buffer

Nuclease-free water c.s. up to 19 ml

T4 ligase (2,000 U / ml) ______________ l_m! _________

Total reaction volume 20 ml

1. Mix gently and centrifuge briefly (5 s) in microcentrifuge.

2. Incubate at room temperature for 2 hours at 16 ° C overnight.

3. Transform each of the ligation reaction mixtures into E. colisupercompetent cells.

20 4. First cool 14 ml round bottom tubes of BD Falcon polypropylene on ice. Prepare SOC medium

up to 42 ° C.

5. Defrost supercompetent cells on ice. When defrosted, mix gently and bring allcuotas of 100 ul of cells into each of the previously cooled tubes.

6. Add 1.7 ml of b-mercaptoethanol to each cell quota. Incubate the cells on ice for 10 minutes, shaking gently every 2 minutes.

7. Add 2 ml of the ligation reaction mixture to an allcuot of cells. Tap the tubes gently.

8. Incubate the tubes on ice for 30 minutes.

9. Pulse heat the tubes in a water bath at 42 ° C for 45 seconds.

10. Incubate the tubes on ice for 2 minutes.

30 11. Add 900 ml of preheated SOC medium and incubate the tubes at 37 ° C for 1 hour with agitation at 225

250 rpm

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12. Sow 20 ml and 200 ml of the transformation mixture on LB agar plates containing carbenicillin.

13. Incubate plates at 37 ° C overnight.

14. Count colonies on the plates and collect 6 colonies for sequencing and PCR testing.

15. Choose a clone with the correct sequence, prepare plasmid DNA and continue with transfection in 293F cells.

293F Cell Transfection

1. One week before transfection, transfer 293F cells to monolayer culture in Dulbecco-modified Eagle medium (D-MEM) supplemented with serum.

2. One day before transfection, plating 0.1 x 105 cells in 100 μl of D-MEM supplemented with serum per transfection sample in 96-well formats.

3. For each transfection sample, prepare DNA-Lipofectamine complexes.

4. Dilute 0.2 mg of DNA in 50 ml of Opti-MEM reduced serum medium. Mix gently.

5. Dilute 0.125 ml of Lipofectamine in 50 ml of Opti-MEM reduced serum medium. Mix gently and incubate for 5 min at room temperature.

6. Combine the diluted DNA with the diluted Lipofectamine. Mix gently and incubate for 20 min at room temperature.

7. Add 100 ml of DNA-Lipofectamine complexes to each well containing cells and medium. Mix gently by shaking the plate back and forth.

8. Incubate the cells at 37 ° C in an incubator with 5% CO2.

9. Add 100 ml of D-MEM supplemented with serum to each well after 6 hours. Incubate the cells at 37 ° C in an incubator with 5% CO2 overnight.

10. Remove the medium in each well by aspiration. Wash each well with 100 ml of 293 SFM II with 4 mM L-glutamine. Add 100 ml of 293 SFM II with 4 mM L-glutamine to each well.

11. Collect the supernatant for ELISA at 96 hours after transfection.

Appendix 1.1: Buffer Formulas

1 x PBS with 2% goat serum

• 2 ml is goat serum

• 98 ml of 1 x PBS

50X TAE buffer

• 242 g of Tris base

• 57.1 ml of glacial acetic acid

• 37.2 g of Na2EDTA-2H2O

• Add distilled H2O to the final volume of 1 liter 1X TAE buffer

• 20 ml of 50X TAE buffer

• 800 ml of distilled H2O

0.8% agarose gel with ethidium bromide

• 0.8 g of LE agarose

• 100 ml of 1X TAE buffer

• Melt the agarose in a microwave oven and stir to ensure uniform mixing

• Cool the agarose up to 55 ° C

5 • Add 2.5 ml of ethidium bromide 20 mg / ml to agarose

• Pour on a gel platform

1% agarose gel with ethidium bromide

• 1 g of LE agarose

• 100 ml of 1X TAE buffer

10 • Melt the agarose in a microwave oven and stir to ensure uniform mixing

• Cool the agarose up to 55 ° C

• Add 2.5 ml of ethidium bromide 20 mg / ml to agarose

• Pour on a gel platform

LB

15 • 10 g of NaCl

• 10 g of tryptone

• 5 g of yeast extract

• Add distilled H2O to a final volume of 1 liter

• Adjust the pH to 7.0 with 5 N NaOH

20 • Autoclave

LB-carbenicillin agar

• 10 g of NaCl

• 10 g of tryptone

• 5 g of yeast extract

25 • 20 g agar

• Add distilled H2O to a final volume of 1 liter

• Adjust the pH to 7.0 with 5 N NaOH

• Submit to autoclave

• Cool to 55 ° C

30 • Add 10 ml of carbenicillin 10 mg / ml sterilized by filtration

• Pour into Petri dishes (25 ml / 100 mm plates)

SOC medium

• 0.5 g of NaCl

• 20 g of tryptone

35 • 0.5 g of yeast extract

• 2 ml of 20% glucose sterilized by filtration

• Add distilled H2O to a final volume of 1 liter

• Submit to autoclave

• Add 10 ml of sterilized 1 M MgCh by filtration and 10 ml of sterilized 1 M MgSOs before filtration

40 use

Washing solution

• 0.05% Tween-20 in PBS Blocking Solution

• 2% Carnation skim milk in PBS

45 Heat inactivated fetal bovine serum

• 500 ml of heat-inactivated fetal bovine serum in the seller's original bottle

• Heat for 30 minutes at 56 ° C with mixing every 5 minutes

• Prepare 50 ml allcuotas and store at -20 ° C

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Dulbecco modified Eagle medium supplemented with serum

• 500 ml of Eagle medium modified by Dulbecco

• 50 ml of heat-inactivated fetal bovine serum

• 5 ml of non-essential amino acids-10 mM MEM

293 SFM II with 4 mM L-glutamine

• 500 ml of SFM II

• 10 ml of 200 mM L-glutamine

DEAE-dextran solution

• 1 g of DEAE-dextran (diethylaminoethyl-dextran)

• Dissolve in 100 ml of distilled water

• Sterilize with filter

Appendix 1.2: Construction of a fully human antibody library

All V regions of kappa light chain (Vk) and heavy chain (Vh) of functional human germline can be obtained from the Vbase database (
http: //vbase.mrc-cpe.cam.ac.u/) and align. The alignments with respect to diversity can then be analyzed, especially in the three framework regions. Then the desired number of Vh and Vk genes can be selected from the resulting sequence clusters for the library construction.

Cloning of region V

Heavy and light chain region V genes (including lattice regions 1, 2 and 3 and CDR1, CDR2 and CDR3) are amplified from human genomic DNA in two fragments using gene specific primers. Then partial genes of region V are combined by overlapping PCR. A linker is added to the V regions of full length LC by nested PCR before cloning into a mammalian expression vector. The sequence of the cloned LC variable domains is confirmed (producing LC clones V). The variable domains of the heavy chain are cloned by TOPO and the sequence is confirmed (HC clones V by TOPO). The HC regions V of the confirmed sequence are amplified from the corresponding plasmids, a linker is added to the 3 'end and the resulting PCR products are cloned in the LC variable domain clones V of LC to form Vk / VH combinations and in A mammal vector.

Full length IgG expression

The expression of IgG 1 heavy chain and full length layer light chain in the desired mammalian vector can be directed from a single promoter, for example, a CMV promoter. Each chain is preceded by a secretion signal that directs the nascent polypeptide chain to the endoplasmic reticulum (ER). An anchor signal can be fused to the C-terminal end of the heavy chain. This signal is cleaved and replaced by an anchor that joins the full-length IgG to the outside of the cell membrane after secretion.

Appendix 1.3: Generation of stable fully human antibody library in mammalian cells by transfection

1. One week before transfection, transfer CHO-S cells to monolayer culture in Dulbecco-modified Eagle medium (D-MEM) supplemented with serum.

2. One day before transfection, sow 6 x 106 cells in 15 ml of D-MEM supplemented with serum per transfection sample in a 10 cm tissue culture plate. Prepare ten 10 cm plates.

3. For each 10 cm plate, prepare DNA-Lipofectamine complexes following steps 4-7.

4. Dilute 25 mg of Maxi-prep fully human antibody library plasmid DNA in 1.5 ml of Opti-MEM reduced serum medium. Mix gently.

5. Dilute 60 ml of Lipofectamine in 1.5 ml of Opti-MEM reduced serum medium. Mix gently and incubate for 5 min at room temperature.

6. Combine the diluted DNA with the diluted Lipofectamine. Mix gently and incubate for 20 min at

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room temperature.

7. Add the 3 ml of DNA-Lipofectamine complexes to each plate containing cells and medium. Mix gently by shaking the plate back and forth.

8. Incubate the cells at 37 ° C in an incubator with 5% CO2 overnight.

9. Change the medium of each plate with 15 ml of D-MEM supplemented with serum. Incubate the cells at 37 ° C in an incubator with 5% CO2 for 48 hours.

10. Detach the cells with detachin detachment medium and resuspend the cells in D-MEM supplemented with serum.

11. Plating 0.4 x 106 cells in 10 ml of D-MEM supplemented with serum with G418 800 mg / ml in a 10 cm tissue culture plate. Transfer all transfected cells resulting in 150 10 cm plates.

12. Plating 0.4 x 106 non-transfected CHO-S cells in 10 ml of D-MEM supplemented with serum with G418 800 mg / ml in a 10 cm tissue culture plate.

13. Feed the cells with D-MEM supplemented with serum with G418 800 mg / ml every 4 days.

14. After 14 days, inspect the plates with untransfected CHO-S cells. There should be no live cells on the plates.

15. Detach the remaining transfected cells with Detachin cell detachment medium and freeze the cells in freezing media at 1x107 8 9 10 cells / ml.

Appendix 1.4: Generation of stable fully human antibody library in mammalian cells by retroviral infection

1. One day before transfection, sow 6 x 106 EcoPack-2 293 cells in 15 ml of D-MEM supplemented with serum per transfection sample in a 10 cm tissue culture plate. Prepare ten 10 cm plates.

2. Prepare the medium containing MBS. This is done immediately before the transfection. For each 10 cm tissue culture plate, 12 ml of medium containing MBS should be prepared.

3. Add 12 ml of medium containing MBS to each 10 cm plate and return the plates to the incubator at 37 ° C. This should be done 20-30 minutes before the addition of the DNA suspension.

4. Resuspend 10 mg of Maxiprep fully human antibody library plasmid DNA sediment in 450 ml of sterile H2O and transfer the DNA to separate 5 ml BD Falcon polystyrene round bottom tubes.

5. Add 50 ml of solution I and 500 ml of solution II of Stratagene Transfection MBS mammal transfection kit to DNA.

6. Gently resuspend any precipitate in the DNA suspension by pipetting the suspension up and down with a pipette set at 500 ml.

7. Incubate the DNA suspension at room temperature for 10 minutes.

8. Remove the 10 cm plates to be transfected from the incubator and add the DNA suspension onto the plates in a dropwise manner, gently shaking to prevent the cells from climbing up the plate and to distribute the DNA suspension evenly.

9. Return the tissue culture plates to the incubator at 37 ° C.

10. After incubating for 3 hours, remove the medium from the plates and replace it with 4 ml of D-MEM supplemented with serum supplemented with 25 mM chloroquine. Return the plates to the incubator at 37 ° C.

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11. After incubating for an additional 6-7 hours, remove the growth medium containing 25 mM chloroquine and replace it with 4 ml of D-MEM supplemented with serum without chloroquine.

12. Incubate the plate in the incubator at 37 ° C overnight.

13. Remove the medium from the plates and replace it with 3.0 ml of D-MEM supplemented with fresh serum. Return the plates to the incubator at 37 ° C.

14. Remove the virus-producing packaging cells from the incubator.

15. Collect the supernatant containing virus from the first plate and filter it through a 0.45 mm filter into a 50 ml sterile conical tube.

16. Bring allcuotas of the viral supernatant into 1.5 ml cryovirus and rapidly freeze in a dry ice / ethanol bath. Store the viral supernatant at -80 ° C.

17. Plating 0.5 x 106 NIH-3T3 cells in 10 ml of D-MEM supplemented with serum on a 10 cm tissue culture plate. Sow 102 10 cm tissue culture plates.

18. Quickly defrost the supernatant by rapid agitation in a H2O bath at 37 ° C.

19. Dilute the virus in DMEM supplemented with calf serum with DEAE-dextran solution with the titer of 0.3 x 105 viral particles / ml. Prepare 3 ml of diluted virus per 100 mm plate to be infected (20% of the cells will be infected). Prepare “simulated cocktail” of growth medium plus DEAE-dextran to be used as a negative control.

20. Remove the medium from the NIH-3T3 cells by aspiration. For each plate, spread 3.0 ml uniformly diluted virus over the cells. Return the plates to the incubator at 37 ° C for 3 hours.

21. After incubation for 3 hours, add an additional 7.0 ml of D-MEM supplemented with calf serum to each plate and return the plates to the incubator at 37 ° C. Incubate the plate for 48 hours.

22. Replace the medium with 10 ml of D-MEM supplemented with calf serum with G418 800 mg / ml in each 10 cm tissue culture plate.

23. Feed the cells with D-MEM supplemented with calf serum with G418 800 mg / ml every 4 days.

24. After 14 days, inspect the plates with simulated infected NIH-3T3 cells. There should be no live cells on the plate.

25. Detach the remaining transfected cells with Detachin cell detachment medium and freeze the cells in freezing medium at 1 x 107 cells / ml.

Example 2. Reactions for the evolution of complete position (CPE)

Mutagenesis reaction

A pair of primers (mixture of primers 1 and mixture of primers 2) are designed for each codon to be inserted. The design will depend on the genetic sequence, and sequence analysis databases such as Sequencher (Gene Codes Corporation) or VectorNTI® (Life Technologies) can be used to design the primers. For CPE, a pair of primers is designed for each codon to be mutated. A degenerate target codon (NNK or NNN) is in the center, flanked by 20 bases on each side (total primer length: 43 bases, clones 9f for sequencing to identify unique mutants). The template DNA is vector DNA with target gene (s).

Prepare the following reactions on 96-well thin wall PCR plates or 0.2 ml thin wall PCR tubes on ice:

Mix of primers 1 (2.5 mM) 5 ml

Mix of primers 2 (2.5 mM) 5 ml

10X DNA polymerase buffer Pfu turbo 2.5 ml

DNA mold (5, 10 25 ng) x ml

dNTP 2 ml

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Nuclease-free water c.s. up to 24.5 ml

Pfu turbo DNA polymerase ________________ 0.5 ml__________________

Total reaction volume 25 ml

1. Prepare a negative control reaction with a 96-well plate (replace primers with TE buffer).

2. Mix gently and centrifuge briefly (5 s) in desktop centrifuge.

3. Cycle the reactions using the cycling parameters explained briefly below:

 Segment

 Cycles Temperature Time

 one

 one

 95 ° C 30 seconds

 2

 18 95 ° C 30 seconds

 55 ° C

 1 minute

 68 ° C

 16 minutes

Quality Control Analysis

1. To submit the amplification reactions to QC, prepare the following reactions in 96-well thin-wall PCR plates or 0.2 ml thin-wall PCR tubes:

Mutagenesis reaction 5 ml

Water 4 ml

Sample loading buffer_____________1 ml

10 ml volume

2. Load 10 ml on a 1% agarose gel -TAE with 0.5 mg / ml ethidium bromide. Use a 1 kb DNA molecular size marker as a standard. Run the gel at 100 V for 20-30 minutes in 1X TAE buffer.

Digest mutagenesis reactions with appropriate restriction enzymes to clone in vector DNA - Example for Dpnl restriction enzyme

1. Add 0.5 ml of the restriction enzyme Dpnl (10 U / ml) directly to each reaction.

2. Mix gently and centrifuge briefly (5 s) in a table top centrifuge.

3. Incubate at 37 ° C in PCR machines for 2 hours.

4. Transform 6 reaction mixtures of each of the 96-well plates into XLI Blue supercompetent cells. Store the rest of the reactions at -20 ° C.

5. Precool 0.2 ml PCR tubes on ice. Heat SOC medium up to 42 ° C.

6. Thaw XLI Blue supercompetent cells on ice. When defrosted, mix gently and bring 50 ml allcuotas of cells into each of the previously cooled tubes.

7. Add 0.8 ml of beta-mercaptoethanol to each cell quota. Incubate the cells on ice for 10 minutes, shaking gently every 2 minutes.

8. Add 2 ml of the reaction mixture to an allcuot of cells. Tap the tubes gently.

9. Incubate the tubes in fried blocks for 30 minutes.

10. Pulse heat the tubes in a water bath at 42 ° C for 45 seconds.

11. Incubate the tubes on ice for 2 minutes.

12. Add 100 ml of preheated SOC medium and incubate the tubes at 37 ° C for 1 hour with shaking at 225250 rpm.

13. Sow the complete transformation mixture on LB agar plates containing carbenicillin.

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14. Incubate plates at 37 ° C overnight.

15. Count colonies on the plates and collect 12 colonies of each transformation reaction for mini preparation and sequencing.

Large scale transformation

1. Thaw XLI Blue supercompetent cells on ice. Defrost 20 competent cell tubes for 96 reactions. When defrosted, add 4 ml of b-mercaptoethanol to each 250 ul tube of competent cells. Incubate the cells on ice for 10 minutes, shaking gently every 2 minutes.

2. Precool 0.2 ml PCR tubes on ice. Heat SOC medium up to 42 ° C.

3. Bring allcuotas of 50 ml of cells into each of the previously cooled tubes.

4. Add 2 ml of the reaction mixture to an allcuot of cells. Tap the tubes gently.

5. Incubate the tubes in fried blocks for 30 minutes.

6. Pulse heat the tubes in a water bath at 42 ° C for 45 seconds.

7. Incubate the tubes on ice for 2 minutes.

8. Add 100 ml of preheated SOC medium and incubate the tubes at 37 ° C for 1 hour with shaking at 225-250 rpm.

9. Sow the complete transformation mixture on LB agar plates containing carbenicillin.

10. Incubate plates at 37 ° C overnight.

11. Grow the cells in 96-well blocks for mini-preparation.

12. Prepare mini-preparation of DNA using the QIAVac 96 kit following the manufacturing protocol.

Example 3. Examine to determine the improvement in antibody affinity

Transfection

One week before transfection, transfer 293F cells to monolayer culture in Dulbecco-modified Eagle medium (D-MEM) supplemented with serum. One day before transfection, see 0.2 x 105 and 0.4 x 105 cells plated in 100 ml of D-MEM supplemented with serum per transfection sample in 96-well formats.

1. For each transfection sample, prepare DNA-Lipofectamine complexes.

2. Dilute 0.2 mg of DNA in 50 ml of reduced serum medium Opti-MEM. Mix gently.

3. Dilute 0.125 ml of Lipofectamine in 50 ml of Opti-MEM reduced serum medium. Mix gently and incubate for 5 min at room temperature.

4. Combine the diluted DNA with the diluted Lipofectamine. Mix gently and incubate for 20 min at room temperature.

5. Add 100 ml of DNA-Lipofectamine complexes to each well containing cells and medium. Mix gently by shaking the plate back and forth.

6. Incubate the cells at 37 ° C in an incubator with 5% CO2.

7. Add 100 ml of D-MEM supplemented with serum to each well after 6 hours. Incubate the cells at 37 ° C in an incubator with 5% CO2 overnight.

8. Remove the medium in each well by aspiration. Wash each well with 100 ml of 293 SFM II with 4 mM L-glutamine. Add 100 ml of 293 SFM II with 4 mM L-glutamine to each well.

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9. Collect the supernatant for ELISA at 96 hours after transfection.

Functional ELISA

1. Coat 96-well Nunc-Immuno Maxisorp plates with 100 ml of 2 mg / ml antigen in coating solution.

2. Cover the plates with sealing elements and incubate overnight at 4 ° C.

3. Decant the plates and gently remove the residual liquid.

4. Add 200 ml of wash solution. Stir at 200 rpm for 5 min at room temperature.

5. Decant the plates and gently remove the residual liquid.

6. Add 200 ml of blocking solution. Stir at 200 rpm for 1 hour at room temperature.

7. Decant the plates and gently remove the residual liquid.

8. Add duplicates of 100 ml / well of control antibody (2 mg / ml) in blocking solution to the plates.

9. Add duplicates of 100 ml of supernatant from the transfection (SOP 5A) to the plates.

10. Stir at 200 rpm for one hour at room temperature.

11. Decant the plates and gently remove the residual liquid.

12. Add 200 mml of wash solution. Stir at 200 rpm for 5 min at room temperature.

13. Repeat steps 11-12 3 times.

14. Add 100 ml of 1: 5000 dilution of purified anti-human goat antibody by affinity conjugated with HRP in blocking solution to each well.

15. Stir at 200 rpm for one hour at room temperature.

16. Decant the plates and gently remove the residual liquid.

17. Add 200 ml of wash solution. Stir at 200 rpm for 5 min at room temperature.

18. Repeat steps 17-18 3 times.

19. Add 100 ml of Sigma TMB substrate to each well. Incubate at room temperature and check every 2-5 minutes.

20. Add 100 ml of 1 H HCl to stop the reaction.

21. Read at 450 nm.

ELISA quantification

1. Coat 96-well Nunc-Immuno Maxisorp plates with 100 ml of Fc-specific human anti-IgG goat antibody purified by affinity 10 mg / ml in coating solution.

2. Cover the plates with sealing elements and incubate overnight at 4 ° C.

3. Decant the plates and gently remove the residual liquid.

4. Add 200 ml of wash solution. Stir at 200 rpm for 5 min at room temperature.

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5. Decant the plates and gently remove the residual liquid.

6. Add 200 ml of blocking solution. Stir at 200 rpm for 1 hour at room temperature.

7. Decant the plates and gently remove the residual liquid.

8. Add duplicates of 100 pl / well of normalized concentration of purified human serum IgG in blocking solution to the plates.

9. Add duplicates of 100 pl of supernatant from the transfection (SOP 5A) to the plates.

10. Stir at 200 rpm for one hour at room temperature.

11. Decant the plates and gently remove the residual liquid.

12. Add 200 pl of wash solution. Stir at 200 rpm for 5 min at room temperature.

13. Repeat steps 11-12 3 times.

14. Add 100 µl of 1: 5000 dilution of affinity purified anti-human goat antibody conjugated with HRP in blocking solution to each well.

15. Stir at 200 rpm for one hour at room temperature.

16. Decant the plates and gently remove the residual liquid.

17. Add 200 pl of wash solution. Stir at 200 rpm for 5 min at room temperature.

18. Repeat steps 17-18 3 times.

19. Add 100 µl of Sigma TMB substrate to each well. Incubate at room temperature and check every 2-5 minutes.

20. Add 100 pl of 1 N HCl to stop the reaction.

21. Read at 450 nm.

Example 4. Generation and examination of a library of Fc codon variants for optimal antibody expression

The present example provides methods to generate a library of Fc codon variants and test methods to obtain Fc variants optimized for enhanced expression in production host cells compared to the parental form of the Fc polypeptide.

A. Design and construction of a library of Fc codon variants

For each codon in the target zone (in this case the Fc part of the human IgG1 molecule) a pair of degenerated primers (direct and reverse) is designed that includes the target codon and 20 bases on each side. The 3rd position of the target codon (hesitation position) contains mixed bases (table 3) that allow the generation of all silent mutations in the target position using the same codon (example A). A second set of degenerate primers is designed for the same codon position if the corresponding amino acid can be encoded by another codon (example B). The corresponding direct and inverse degenerate primers are mixed 1: 1, paired with the mold and spread to obtain full length products by strand displacement using a thermostable DNA polymerase. The mold is digested with Dpnl and the full length extension products are transformed into E. coli. Up to 12 colonies are sequenced by mutagenesis reaction. Confirmed mutants are arranged for sequence in 96-well plates and stored in glycerol. Glycerol reserves are used to perform plasmid DNA minipreparations for transfection in mammalian cells and their examination.

Table 3: Codes for degenerate bases in synthetic oligonucleotides

 Slbolo

 Mixed base

 R

 A, G

 Y

 C, T

 M

 A, C

 K

 G, T

 S

 C, G

 W

 A, T

 H

 A, C, T

 B

 C, G, T

 V

 A, C, G

 D

 A, G, T

 N

 A, C, G, T

Example A: target codon = CCC (proline)

^ ■ direct primer: CCD, reverse primer: HGG Example B: target codon = TCG (serine)

5 ^ direct primer 1: TCH, reverse primer 1: DGA

^ direct primer 2: AGY, reverse primer 2: RCT

The 20 bases flanking the target codon are not shown. Total primer length: 43 bases.

B. ELISA-based expression and examination of the Fc codon variant library

Clones from the Fc codon variant library were transfected into a mammalian cell line. Full length IgGs were produced and secreted into the medium. Supernatants of the expressed Fc codon variants were examined to determine the level of IgG expression higher than that of the parental clone using ELISA assay. ELISA data was normalized with a beta-galactosidase assay that measures transfection efficiency. The best coincidences identified in the primary exam were transfected again and examined three times to confirm the increased expression level.

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Claims (15)

10 fifteen twenty 25 30 35 1. Method of evolution and expression of an antibody in a eukaryotic cell production host; Understanding the method: to. select a template antibody; b. evolve the template antibody to allow the production of a set of mutant antibodies in a eukaryotic cell production host; C. examine the mutant antibodies to determine at least one predetermined property, characteristic or activity; d. selecting a higher mutant antibody from the set of mutant antibodies based on the optimization of the at least one predetermined property, characteristic or activity compared to the same property, characteristic or activity of the template antibody; Y and. express the higher mutant antibody in the same eukaryotic cell production host as in step (b) for any commercial scale. 2. Method according to claim 1, wherein the selection stage (a) comprises: a1. generate a library of anti-antigen antibodies in a eukaryotic cell production host with presentation on the cell surface of antibodies; a2. examine the library to determine at least one predetermined property, characteristic or activity; and a3. Select a template antibody from the library. 3. Method according to claim 1 wherein the eukaryotic cell production host is selected from 3T3 mouse fibroblast cells; BHK21 Syrian Hamster Fibroblast Cells; dog epithelial cells, MDCK; Hela human epithelial cells; Kangaroo rat epithelial cells PtK1; SP2 / 0 mouse plasma cells; and NS0 mouse plasma cells; human embryonic kidney cells HEK 293; monkey monkey kidney cells; Chinese hamster ovary cells CHO, CHO-S; R1 mouse embryonic cells; E14.1 mouse embryonic cells; human embryonic cells H1; human embryonic cells H9; human embryonic cells PER C.6; S. cerevisiae yeast cells; and Picchia yeast cells. 4. Method according to claim 3, wherein the eukaryotic cell production host is CHO or HEK293. 5. Method according to claim 1, wherein the examination steps comprise fluorescence activated cell classification (FACS). 6. Method according to claim 1, wherein the step of evolving comprises producing a set of mutant antibodies formed from the template antibody having m complementarity determining regions (CDR), wherein m is an integer number of 1 to 6, each of said CDRs comprising n amino acid residues, the method comprising: to. generate m x n independent sets of antibodies, each set comprising member antibodies that have a predetermined number of different amino acid residues at a predetermined individual position of the CDR; in which each set of antibodies differs in the predetermined individual position; and the number of different member antibodies generated is equivalent to m x n x X. 7. Method according to claim 6 wherein m is 6. 8. Method according to claim 1 wherein the template antibody is an antibody fragment selected from a heavy chain, light chain, variable domain, constant domain, hypervariable region, complementarity determining region 1 (CDR1), determining region of the Complementarity 2 (CDR2) and determining region of Complementarity 3 (CDR3). 9. Method according to claim 1, wherein the step of evolving (b) comprises h. generate n-1 independent sets of mutant polypeptides from the template antibody, each set comprising member polypeptides having an X number of amino acid residues 5 10 fifteen twenty 25 30 35 different presets in a predetermined individual position of the polypeptide; in which each set of polypeptides differs in the predetermined individual position; and the number of different member polypeptides generated is equivalent to [n-1] x X; and in which the exam stage (c) comprises: i. test each member polypeptide to determine the at least one predetermined property, characteristic or activity; j. identify any change in said property, characteristic or activity of the member polypeptide in relation to the template antibody; k. create a functional map in which the functional map is used to identify positions and mutations in the mutant polypeptide that result in a superior mutant and / or a silent mutation compared to the template antibody. 10. Method according to claim 9, wherein X is 19. 11. Method according to claim 10, wherein said generation step comprises: i. subjecting a polynucleotide containing codons encoding said template antibody to polymerase-based amplification using a degenerate oligonucleotide 64 times for each codon to be mutagenized, in which each of said degenerate oligonucleotides 64 times is composed of a first homologous sequence and a triplet sequence N, N, N degenerate, so that a set of progeny polynucleotides is generated; Y ii. subjecting said set of progeny polynucleotides to clonal amplification so that mutant polypeptides encoded by the progeny polynucleotides are expressed. 12. Method according to claim 1, wherein the property, characteristic or predetermined activity is selected from reduction of antibody-protein aggregation, enhancement of antibody stability, increase in antibody solubility, introduction of glycosylation sites, introduction of conjugation sites, reduction of immunogenicity, potentiation of protein expression, increase in antigenic affinity, decrease in antigenic affinity, change in binding affinity, change in immunogenicity and potentiation of specificity. 13. Method according to claim 2, wherein the library of anti-antigen antibodies is a library of humanized anti-antigen antibodies. 14. Method according to claim 1, wherein the selection step (d) comprises: selecting the upper mutant antibody based on an improved expression compared to the template antibody; Y wherein the expression stage (e) comprises manufacturing the superior mutant antibody in the same eukaryotic cell production host as in the stage of making it evolve (b). 15. Method according to claim 1 wherein the step of evolving (b) comprises one of complete position evolution (CPE); full position insertion evolution (CPI); complete position deletion evolution (CPD); complete position evolution (CpE) followed by combinatorial protein synthesis (CPS); complete position deletion evolution (CPD) followed by combinatorial protein synthesis (CPS); or evolution of complete position deletion (CPD) followed by combinatorial synthesis of proteins (CPS).